Ultrastructural labeling of lymphocyte surface immunoglobulin: the preparation and use of soluble immune complexes as indirect immunoelectromicroscopic markers.

2 distinct macronuclear markers, ferritin and hemocyanin, may be used in a mixed anti-globulin labeling reaction to localize lymphocyte surface immunoglobulin (Ig) determinants by transmission electron microscopy. Soluble immune complexes of the marker molecules (antigen) are prepared by complexing with specific antiserum in 40 to 50 x antigen excess; uncomplexed Ig is removed by ultrascentrifugation and/or gel filtration chromatography. Immunoelectrophoresis, spectrophotometry and passive hemagglutination inhibition assay are used to determine the purity and amounts of antibody-antigen in the purified immune complexes. For immunoelectron microscopic labeling, the immune complex markers are coupled to lymphocyte surface Ig by an indirect anti-Ig or anti-allotype antibody linkage. Labelled Ig determinants at 0 degrees C or in the presence of sodium azide are visualized as small patches of marker molecules on the lymphocyte surface membrane. This EM labeling method results in much more consistent and generally higher percentages of surface Ig positive cells (60 to 70% of rabbit peripheral blood lymphocytes) than the percentages obtained using other methods, such as immunofluorescence or autoradiography. If the lymphocytes are warmed to 37 degrees C in the absence of azide the labeled surface Ig determinants undergo rapid endocytosis; endocytotic vesicles containing marker molecules are visible. This mixed anti-globulin immunoelectronmicroscopic labeling system may be used to localize a wide variety of antigens on different cell surfaces.     

The use of the preparation of F(ab')2 antibody from soluble immune complexes to determine the complexed antigens.

An autoradiographic technique for the characterization of antibody specificity in soluble antigen--antibody complexes has been developed. The circulating antigen--antibody complexes are precipitated by polyethylene glycol (PEG). The antibodies are liberated as F(ab')2 from the complexes by pepsin digestion. The antibody specificity against the putative antigen was revealed by radioimmunoelectrophoresis using [125I]-F(ab')2 reagents. The method was developed by using an experimental model of C3/anti-C3 complexes.     

An international collaborative study to assess a set of reference reagents for HIV-1 PCR.

An international collaborative study was performed to evaluate a set of PCR reference reagents for HIV diagnosis. Twenty-six laboratories from 9 countries analysed a proficiency panel of 10 coded DNA samples using the PCR reference reagents and protocols. For comparison, these coded samples were then assessed using a laboratory's own 'in-house' reagents and methodologies. The objectives of the study were: (i) to assess inter-laboratory variation of PCR sensitivity, (ii) to evaluate the DNA 'carryover' problem and frequency of false negative results and (iii) to examine the utility of the complete set of reagents and templates to act as reference preparations for HIV PCR. Using the reference reagents, 46% of laboratories reported no false positive results in any of their assays of the negative controls. The remaining laboratories all reported a false positive result(s) in at least one assay. The overall false positive result rate for the study was 9.3%. In contrast, an overall false negative result rate of 7.4% was observed, with some laboratories recording negative results even for samples containing 10,000 molecules of target DNA. The level of absolute sensitivity may be assessed accurately only from the 12 laboratories that obtained no false positive results. All 12 laboratories detected the sample containing 10 molecules of template DNA and 9 out of the 12 laboratories detected the sample containing 1 molecule. This is in close agreement with the theoretical detection rate based on a statistical probability model for the detection of a single molecule. These characterised reference reagents were at least as sensitive as any of the 'in-house' reagents and methodologies applied, including nested PCR. The complete set of characterised reference reagents is now available for quality control assessment of HIV-1 PCR from the MRC ADP.     

Enzyme/anti-enzyme monoclonal antibody soluble immune complexes (EMAC): their use in quantitative immunoenzymatic assays

Enzyme/anti-enzyme antibody soluble immune complexes were prepared with monoclonal mouse antibodies (MA) which were directed against peroxidase (PO) and beta-galactosidase (GAL). These enzyme monoclonal antibody complexes (EMAC) functioned as markers to quantify mouse antibodies using an enzyme immunoassay which incorporated an anti-mouse Ig as the bridge between the EMAC and the specific antibody bound to an antigen immobilized on a polystyrene plate. EMAC prepared with PO (PO-MAC) or with GAL (GAL-MAC) were both effective in quantifying polyclonal as well as monoclonal mouse antibodies, and gave sensitivity equal or superior to that obtained with covalent enzyme/anti-mouse Ig conjugates. The smaller amount of antibodies detected with EMAC depended on the affinity of both the antibody tested and the monoclonal antibody used to prepare EMAC. This method is an improvement on the 'antibody bridge' method, because EMAC can be prepared easily by simply mixing the enzyme and the MA at the appropriate amounts 2 h prior to use. In addition, EMAC can be prepared using crude preparations of enzyme and unpurified ascitic fluids containing the MA, thus decreasing the cost of the test considerably.     

Results of IVF from a prospective multicentre study.

Part of a cost-effectiveness study of in-vitro fertilization was the evaluation of the medical results of this fertility treatment. Data were prospectively collected from greater than 3000 IVF treatments in five Dutch hospitals during a 2-year period. The average 'take-at-least-one-healthy-baby-home-rate' per started treatment was 10% (the average clinical pregnancy rate per embryo transfer was 20%). After more IVF treatments, about one in three to four couples were successful. Differences in results were mainly caused by patient characteristics, the treatment episode and the treating hospital. These differences remained in a multivariate logistic regression analysis.     

Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study.

BACKGROUND: A number of international research groups have developed DNA quantitation assays in order to investigate the role of mitochondrial DNA depletion in anti-retroviral therapy-induced toxicities. OBJECTIVES: A collaborative study was undertaken to evaluate intra-assay precision and between laboratory concordance of measurements of mitochondrial DNA quantity, as a component of a comprehensive quality assurance project. STUDY DESIGN: Four laboratories were asked to measure and report mitochondrial DNA and nuclear DNA genome copy number, as well as mitochondrial DNA copy number/cell, for 17 coded aliquots of DNA derived from serial dilutions of pooled DNA from a lymphoblastoid cell line. Samples included masked replicates and five standards. All samples had similar mitochondrial DNA/nuclear DNA ratios. Precision within laboratories was assessed by determining the coefficient of variation of replicates. Concordance between laboratories was assessed by determining the average coefficient of variation of the mean replicate values for each sample. The effect of standardising the assay for these three measurements was also assessed for laboratories A, B and C. RESULTS: Measurements of mitochondrial DNA and nuclear DNA content for replicate samples varied by an average of less than 6% (based on log(10) values, 72% non-logged values), and measurements of mitochondrial DNA/cell for replicates varied by less than 12% (based on log(10) values, 32% non-logged values), with no improvement of precision after standardisation. Standardisation did significantly improve the concordance of results for measurements of mitochondrial DNA content and mitochondrial DNA/cell. Non-standardised measurements of mitochondrial DNA content for the same sample set varied by 19% between laboratories (based on log(10) values, 96% non-logged values), and after standardisation results varied by less than 3% (based on log(10) values, 54% non-logged values). There was no significant improvement for concordance of measures of nuclear DNA content after standardisation, with results varying by 4.56% between laboratories (based on log(10) values, 45% non-logged values) before standardisation, and by 2.49% (based on log(10) values, 50% non-logged values) after standardisation. Derived values of mitochondrial DNA/cell varied between laboratories by an average of 91% (non-logged, 56% log(10) values) before and by 56% (non-logged, 13% log(10) values) after standardisation. CONCLUSION: All assays demonstrated good precision. The use of common standards is an important step in improving the comparability of data between laboratories.     

Placental barrier against circulating immune complexes. A study in pregnant guinea pigs using maternal injection of horseradish peroxidase soluble immune complexes.

It is not rare that the so-called immune complex diseases (some of which were recently supposed to be induced by the biological effects of circulating antigen-antibody immune complexes) are concomitantly present in pregnant females. But there have been very few reports of congenital immune complex diseases.     

Antigen-specific detection of soluble immune complexes in conglutinin-binding assays.

A modification of the conglutinin-binding assay has been used for antigen identification in circulating immune complexes (IC). The complexed antigen was identified, in conglutinin-bound complexes, by antibodies or antibody fragments, specifically directed against the antigen. These antibody preparations were either radiolabelled or detected with 125I-protein A. This system was found efficient using in vitro-formed bovine serum albumin--anti-bovine serum albumin and tetanus toxoid--anti-tetanus toxoid soluble immune complexes at equivalence or at slight antigen or antibody excess. In addition, with 125I-IgG--anti-HBs and with 125I-F(ab')2--anti-HBs, we examined 44 sera from 41 patients with viral type B hepatitis and the presence of HBs was observed in 18 (40.9%) IC containing tested samples. Therefore, this method appears applicable to clinical investigation.     

Results of the first World Health Organization international collaborative study of detection of human papillomavirus DNA

Twenty-nine laboratories in 12 countries participated in a study to assess the performance of various human papillomavirus (HPV) detection assays through the use of a recombinant HPV DNA standard reagent panel. The panel was designed by a group of HPV experts, and samples were prepared and distributed by the World Health Organization International Laboratory for Standards and Biologicals in The Netherlands. Each panel consisted of 24 coded samples including a dilution series for HPV types 16 and 18, alone or in combination with five other high-risk (HR) HPV types including HPV types 31, 33, 35, 45, and 52, the low-risk HPV type 6, and a negative control. Qualitative assays were generally consistent across laboratories, and most invalid results reflected a lack of HPV test sensitivity. The combined data sets had a proficiency for HPV 16 of 62.5% (15/24) and for HPV 18 of 73.9% (17/23). HPV 31 was the least accurately detected by participating laboratories. Approximately half of participating laboratories failed to detect high concentrations of HPV 31 and, to a lesser extent, to detect HPV types 35, 52, and 6. The panel sample materials offer a source of renewable and reproducible material that could be used in the future development of international standard reagents for calibration of HPV DNA assays and kits.     

Antigen-specific detection of soluble immune complexes by a solid phase specific antibody system.

An antigen-specific immune complex assay based on the following principle has been developed: xenogeneic, antigen-specific antibodies are attached to a solid phase. During first incubation with patient's serum, immune complexes in antigen excess are bound to the xenogeneic antibody by their free antigenic determinants. In a second incubation a labeled antibody, specific for human immunoglobulins, is combined with the antibody part of the immune complex. Quantitation of the label allows the determination of the immune complexes. The principle of the method has been varied using artificial soluble immune complexes of tetanus toxoid and human anti-tetanus toxoid antibody, and immune complexes prepared by mixing patient sera containing either HBsAg or anti-HBsAg antibodies. The reliability of the results is shown by their coefficient of variation (2.5%). With the method described soluble immune complexes predominantly in slight antigen excess, which are thought to be responsible for development of immune complex disease, have been detected.     

Quantitation of human IgG subclass antibodies to Haemophilus influenzae type b capsular polysaccharide. Results of an international collaborative study using enzyme immunoassay methodology.

An international collaborative study was conducted at ten sites to examine the performance of enzyme immunoassays (EIAs) for the quantitation of IgG1, IgG2, IgG3, IgG4 and total IgG anti-Haemophilus influenzae type b (Hib) capsular polysaccharide in human serum. All groups used the same reagents: microtiter plates coated with polyribosylribitol phosphate (PRP) conjugated to poly-L-lysine (PLL), reference, control and test human sera, biotin-conjugated International Union of Immunological Societies (IUIS)-documented monoclonal anti-human IgG1-4 and IgG Pan detection antibodies, avidin-peroxidase and TMB substrate. Initial mixing of soluble PRP antigen or an equal volume of buffer with the 20 test sera prior to analysis confirmed PRP antigen specificity in all five EIAs with greater than 80% competitive inhibition at most sites. Positive correlation between the total IgG anti-Hib and sum of IgG1-4 anti-Hib was demonstrated (r2 = 0.99, Y = 1.13X -0.15). Good agreement was shown between the total IgG anti-Hib as measured by EIA and the total Hib-specific antibodies measured by the current radiolabeled antigen binding assay (r2 = 0.97, Y = 4.6X -5.8). Assay parallelism was demonstrated with an average interdilutional %CV of 22% and parallel dose-response curve slopes. The interdilutional %CVs were calculated as an average per sample of the variation of microgram/ml (corrected for dilution) at different dilutions per laboratory for all participating sites. The interlaboratory variation was the only performance parameter studied that exceeded the target level of 35% CV in all IgG1-4 and total IgG anti-Hib assays. IgG subclass distributions in the test sera demonstrated a predominance of IgG1 anti-Hib in the pediatric serum pools and IgG2 anti-Hib in the adult sera, with low but detectable levels of IgG3 and IgG4 anti-Hib in each group.     

Detection of circulating soluble immune complexes in patients with various renal diseases.

Sera from patients with various types of glomerulonephritis (GN) as well as sera from rabbits with acute serum sickness were studied for the presence of circulating immune complexes (IC). The method used is based on the observation that IC inhibit the uptake of IgG aggregates by guinea-pig peritoneal macrophages. Inhibition significantly greater than with normal human sera was found with sera of patients with membranous GN, membranoproliferative GN, focal glomerular sclerosis, minimal change nephrotic syndrome, acute septicaemic glomerular diseases and systemic lupus erythematous. IC were also detected in rabbits with acute serum sickness during the period of immune elimination.     

An international collaborative study to establish the 1st international standard for HIV-1 RNA for use in nucleic acid-based techniques

Twenty-six laboratories from 10 different countries participated in a collaborative study to establish the 1st International Standard for HIV-1 RNA for use in nucleic acid-based techniques (NAT). Three candidate preparations were tested all based on genotype B viruses. The candidates were tested by each laboratory at a range of dilutions in four independent assays and the results collated and analysed statistically. All three candidates gave results that were tightly grouped, with little difference between the results from different laboratories or from the use of different assays. Studies of relative potency showed good agreement between laboratories. There were no significant differences between five commercial assay types, except that candidate XX showed a slightly lower potency compared to YY and ZZ with a single commercial assay. The reason for this was not established. Degradation studies showed that the freeze-dried preparations were stable at -20,4 and 20 degrees C for 26 weeks, the longest period studied, but that they became difficult to reconstitute after 3 weeks at 45 degrees C and 9 weeks at 37 degrees C. As a result of the study, the World Health Organisation (WHO) Expert Committee on Biological Standardisation (ECBS) established the preparation referred to as candidate YY (NIBSC Code No. 97/656) as the 1st International Standard for HIV-1 RNA for use with NAT with an assigned potency of 100000 International Units per vial.     

The effect of lidocaine on the processing of soluble immune aggregates and immune complexes by peritoneal macrophages.

In a search for new techniques which would specifically identify the various steps of the handling of immune complexes by macrophages the local anaesthetic, lidocaine, was investigated. Lidocaine was shown to inhibit selectively the digestion of immune complexes and immunoglobulin aggregates by macrophages whereas it had little effect on the binding or ingestion steps. Due to the reversible inhibitory effect of lidocaine, this drug may be used in future studies to assess the role of extracellular stimuli on the fate of intracellular immune complexes.     

A quantitative approach to the determination of antigen in immune complexes

Immune complexes are present in the circulation of healthy individuals and the formation of such complexes is part of a normal immune process. During some pathological conditions, significant amounts of immune complexes are formed and deposited in the kidney and other tissues, causing severe injury. Since the levels of immune complexes can provide valuable prognostic information, dozens of methods have been developed to detect and quantify these complexes. However, many of these methods are non-specific, not quantitative, and give false-positive results. Methods based on detecting the antigen portion of immune complexes can yield more precise information about circulating immune complexes. We have used a quantitative dot-blot assay, which permits detection of antigen even if buried, to determine the levels of antigen in circulating immune complexes. In healthy donors, significant amounts of immune complexes containing DNA and beta(2)-glycoprotein I were detected (natural immune complexes). Natural immune complexes with Lewis X antigen were also observed in the circulation of healthy persons. In experimentally induced murine systemic lupus erythematosus (SLE) and SLE patients, there was a correlation between the clinical manifestations and the levels of DNA in the circulating immune complexes. At severe SLE flares, the level of DNA in circulating immune complexes decreased, probably due to tissue deposition of immune complexes. The low levels of DNA in immune complexes circulating in SLE patients correlated with low serum concentrations of the complement component C1q. No direct correlation was found between the levels of circulating anti-dsDNA antibodies and DNA in immune complexes. Thus, quantitation of antigen levels in circulating immune complexes can be used to determine the prognosis of autoimmune diseases.     

Critical events in the irreversible uptake of soluble immune complexes by macrophages

Small, soluble, IgG-containing complexes bind to phagocytes via Fc receptors on the cell membrane, in competition with IgG in the environment. Following attachment, the complexes rapidly rearrange themselves into larger aggregates by forming additional Ag-Ab bridges. The driving force for this rearrangement is a massive increase in the effective concentration of the complexes at the receptor-bearing surface. Aggregation precedes ingestion and probably constitutes the critical irreversible step which predisposes complexes to selective clearance from the circulation.     

Criteria for the standardization of Salmonella mutagenicity tests: results of a collaborative study. III. The influence of the composition and preparation of the minimal medium in the Salmonella mutagenicity test

The influence of factors connected with the preparation of the minimal medium for the Ames test has been investigated. Faulty sterilization procedures can lead to the generation of toxic and/or mutagenic by-products in the minimal medium. Changes in histidine concentration affect not only the number of spontaneously arising colonies on the plate, but also the number of induced mutants. Although the number of spontaneous mutants increases slightly with increasing histidine concentration, the influence on the number of induced mutants depends on the nature of the mutagen tested.     

Enhanced degradation of soluble immune complexes by guinea-pig peritoneal macrophages in the presence of complement.

The role of complement in the processing of soluble immune complexes by guinea-pig peritoneal macrophages was studied in an homologous system in vitro by using immune complexes prepared with bovine thyroglobulin as the antigen and guinea-pig IgG2 antibodies. The simplest complexes showing complement activation and which were degradable by macrophages had a composition of Ag1 Ab2-3. Complement was shown to have an enhancing effect on the degradation of complexes which had an antibody: antigen ratio in the complexes which was at least 4 (Ag1 Ab4). The effect of size on complement activation and degradation of the complexes by macrophages was studied by employing the observation that immune complexes increase in size during their preparation. In the presence of serum as a complement source it was shown that degradation of small complexes by macrophages was inhibited whereas the degradation of large complexes was enhanced. The enhanced degradation of complexes in the presence of fresh serum did not occur in C4-deficient serum nor in EDTA-serum, which indicates that the observed effect is complement mediated. The experiments described here thus extend and confirm earlier studies using heat aggregated immunoglobulins and show that complement may play an important role in the elimination of immune complexes in vivo.