The international standard for interleukin-6 Evaluation in an international collaborative study

Three ampouled preparations of interleukin-6 (IL-6) were evaluated by 12 laboratories in seven countries for their suitability to serve as the international standard of IL-6. The preparations were assayed using in vitro bioassays and immunoassays. On the basis of the results reported here, with the agreement of the participants in the study and with the authorization of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) one of the preparations (coded 89/548) was established as the international standard of IL-6.     

The international collaborative study establishing the first international standard for timothy (Phleum pratense) grass pollen allergenic extract

A selected candidate international reference preparation of timothy grass (Phleum pratense)-pollen extract was studied together with two other freeze-dried timothy pollen allergenic extracts in a multinational study. The collaborators used RAST inhibition, histamine release, quantitative immunoelectrophoresis (crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis and rockets), isoelectric focusing, and other methods. The total allergenic potencies measured in RAST inhibition were evaluated for validity of linearity and parallel-line response. The relative concentrations of some important individual allergenic components were measured. The relative potencies for the total allergenic activity and the timothy components studied in each preparation were expressed relative to the selected candidate. This preparation was established in 1983 by the World Health Organization expert committee on biologic standardization as the international standard for timothy grass-pollen extracts with assigned units of 100,000 IU per ampule.     

The international collaborative study on the first international standard of birch (Betula verrucosa)-pollen extract

A selected candidate international standard preparation of birch (Betula verrucosa)-pollen extract was studied together with other birch-pollen extracts in a multinational study involving 20 laboratories in 11 countries. The biologic activity of the extract had previously been demonstrated in quantitative skin prick testing. The study methods comprised RAST inhibition, histamine release, quantitative immunoelectrophoresis, isoelectric focusing, and other methods. The results from RAST inhibition were calculated as parallel-line assays with statistical tests for linearity and parallelism. Analysis of variance was applied to test the significance of differences between potency estimates. In all assay methods, the candidate standard could be used to assign relative potencies to other birch-pollen extracts. The candidate standard was adequately stable during 36 months of storage at or below 5 degrees C. On the basis of this study, the World Health Organization has established the preparation as the International Standard for birch-pollen extract with assigned units of 100,000 IU per ampule.     

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.     

Implications for the assay and biological activity of interleukin-8: results of a WHO international collaborative study

Eight ampouled preparations of interleukin-8 (IL-8) have been evaluated for their suitability to serve as an international standard for IL-8 by 30 laboratories in 12 countries in an international collaborative study. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. It is clear from the study that different recombinant preparations of IL-8 can have different biological specific activities, even though all were produced using E. coli. It is of interest that the intra-laboratory variability of estimates provided by several neutrophil degranulation bioassays was less than that of the immunoassays, suggesting that these bioassays can be as precise, if not more so, than immunoassays. In addition, immunoassay estimates of IL-8 preparations differed from those of bioassays, illustrating the fact that immunoassays do not necessarily measure biologically active cytokine. The large reduction in the inter-laboratory variability of estimates in terms of a common reference preparation clearly illustrates the need for a standard for IL-8. On the basis of the results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-8 (89/520) was established as the International Standard for IL-8 with an assigned unitage of 1000 IU/ampoule.     

Growth charts for cri-du-chat syndrome: an international collaborative study

Low birth weight and slow growth are frequently observed in the patients with cri-du-chat syndrome. To provide a growth reference standard for children with cri-du-chat syndrome, syndrome-specific growth charts have been developed from a combination of cross-sectional and longitudinal measurements on 374 patients from North America, Italy, Australia, and the British Isles. The data were obtained from pediatric records, parent reporting, and personal examinations at national 5p- parent support group meetings in the U.S., Italy, U.K., and Australia. The growth curves include height and weight measurements for patients ages 0 to 18 years and head circumference measurements for patients ages 0 to 15 years. Birth weight was above the 5th percentile of general population in 50% of cases: mean weight 2.8 kg +/- 1.85 SD for males and 2.6 kg +/- 1.51 SD for females. Growth curve medians were usually at or below the 5th centile of reference populations throughout life. The median head circumference falls below the 2nd centile, and this change increases with age. The charts show that compared with the standard population, most children with cri-du-chat syndrome are small at birth and as they grow most, but not all, have significant microcephaly and compromised weight for age, and to a lesser extent, compromised height for age. Am. J. Med. Genet. 94:153-162, 2000.     

Control of human T-colony formation by interleukin-2.

T-colony formation can be induced in PHA-stimulated peripheral blood mononuclear cells (PBM) from man, but not in PHA-stimulated purified T cells, the latter requiring the presence of factors produced by PHA-stimulated PBM and termed T-colony promoting activity (TCPA). In this paper, we demonstrate that interleukin-2 (IL-2), the growth hormone of T lymphocytes, controls T-colony formation. We show that: IL-2 activity and TCPA produced by PHA-activated PBM are co-purified by gel filtration and chromatography on blue agarose, a procedure which yields a 850-fold IL-2 purification; recombinant IL-2, produced by genetically manipulated Escherichia coli, can induce T-colony formation in PHA-stimulated purified T cells; Monoclonal antibody against the IL-2 receptor (anti-Tac antibody) completely inhibits the T-colony formation in PHA-stimulated PBM when directly added to the culture system.     

An enzyme-immunoassay for human interleukin-2

A highly sensitive enzyme-immunoassay (EIA) for human interleukin-2 (IL-2) has been established. The assay is based on a sandwich method that uses two kinds of anti-IL-2 antibodies raised against Escherichia coli-derived recombinant IL-2 (rIL-2). An affinity-purified-anti-IL-2 goat IgG was used as the first antibody and the Fab' fragment of an affinity-purified-anti-IL-2 rabbit IgG was used as the second antibody after being coupled with horseradish peroxidase (HRP). As little as 30 pg/ml of IL-2 was detected by the EIA, indicating that this method was about 100 times more sensitive than the bioassay using an IL-2-dependent murine natural killer cell line, NKC3. There was a good correlation between the EIA and the bioassay (r = 0.998).     

A collaborative study on the first international standard of Dermatophagoides pteronyssinus (house dust mite) extract

A collaborative study was carried out to assess the suitability of a preparation to serve as the International Standard for Dermatophagoides pteronyssinus (house dust mite) extract. The proposed international standard of D. pteronyssinus, two additional freeze-dried extracts, and a commercially available skin testing solution were tested in the study. Nineteen laboratories in 11 different countries participated. The assay methods used included RAST inhibition, crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis, isoelectric focusing, quantitative skin testing, and various other methods for assessing total allergenic activity. In addition, six laboratories measured the quantity of antigen P1, and three laboratories measured antigen DpX in each of the preparations. On the basis of the results from this study, the World Health Organization established the preparation as the International Standard for D. pteronyssinus extract with an assigned unitage of 100,000 IU per ampule. The units refer both to the total allergenic activity of the ampule and to that of the individual allergens, such as P1 and DpX.     

Direct induction of human B-cell differentiation by recombinant interleukin-2.

Recombinant interleukin-2 (rIL-2) induced highly purified human tonsillar B cells to differentiate into immunoglobulin (Ig)-producing cells in vitro. The B-cell response was not due to rIL-2-contaminating substances, but reflected the activity of IL-2 itself, since it was inhibited by addition to the cultures of anti-TAC monoclonal antibody. The rIL-2-induced B-cell response was apparently not mediated by factors released by residual T cells present in B-cell suspensions at undetectable levels, since supernatants (SN) from unstimulated autologous T cells cultured at concentrations even much higher than those possibly contaminating B-cell suspensions did not induce any detectable Ig production. In addition, the Ig production by B cells cultured with SN prepared from high numbers of autologous T cells stimulated with rIL-2, as well as from allo-activated or mitogen-stimulated T cells, was of the same magnitude as the Ig production resulting from direct addition of rIL-2 concentrations comparable with those present in the supernatants. After centrifugation on Percoll density gradients, most of the tonsillar B cells responsive to rIL-2 were recovered in the lower density cell fraction containing a number of larger activated B cells. Moreover, B-cell enriched suspensions from peripheral blood (PB) (which usually contains a lower number of in vivo activated B cells than tonsil) showed poor or no response to rIL-2 alone, but displayed significant Ig production when rIL-2 was added to the cultures in the presence of Staphylococcus aureus Cowan I (SAC) bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Evaluation of a candidate international standard preparation for human anti-Toxoplasma immunoglobulin G

A freeze-dried human serum preparation containing immunoglobulin G (IgG) to Toxoplasma gondii was assessed for its suitability as an international reference reagent in an international collaborative study by 24 laboratories from 17 countries. This candidate standard was compared with the third international standard (IS) for human anti-Toxoplasma serum, TOXM, with the previous second IS, TOXS, and with a range of other serum samples. Samples were tested with the Sabin-Feldman dye test and a range of agglutination assays and enzyme immunoassays. This study emphasizes the need for appropriate standards if intermethod agreement of estimates is to be achieved. On the basis of the results of this study, the preparation was established by the World Health Organization as the first IS for human anti-Toxoplasma IgG, with an assigned potency of 20 IU per ampoule of total anti-Toxoplasma antibodies.     

Biotinylation of human interleukin-2 for flow cytometry analysis of interleukin-2 receptors

We designed a method to analyze receptors for interleukin-2 (IL-2R) using biotinylated IL-2 (b-IL-2). To optimize the condition of biotinylation of IL-2 for flow cytometry, the degree of biotinylation was controlled by monitoring the relative biotin contents in b-IL-2 with a newly developed ELISA. The b-IL-2 prepared by incubating 150 micrograms IL-2 in 150-300 micrograms/ml N-hydroxysuccinimidyl biotin retained biological activity and was appropriate for flow cytometry analysis. Positive fluorescence appeared in the IL-2R-bearing cell lines but not in those without IL-2R. This binding was inhibited by preincubation of the cells with unlabelled IL-2. The b-IL-2 bound to both low and high affinity IL-2Rs, but the binding to the latter was more intense. The advantage of this method is that expression of IL-2Rs of these two categories of affinity can be separately monitored.     

Inactivation of human interleukin-2 (IL-2) by alpha 2-macroglobulin-trypsin complexes.

The loss of the biological activity of interleukin-2 (IL-2, T-cell growth factor) in the presence of alpha 2-macroglobulin-trypsin (alpha 2M.t) complexes has been investigated using an IL-2-dependent cloned 'cytotoxic' murine T-cell line. While reaction mixtures of native alpha 2M, aprotinin or methylamine-treated alpha 2M and IL-2 had no effect on IL-2 activity when incubated at 37 degrees for 5 h, alpha 2M.t (90 nM, [T]:[alpha 2M] = 0.8) inactivated IL-2 at a rate of one-sixth of that of the free enzyme. This effect was abolished by treatment of alpha 2M.t with aprotinin (MW 6500). Soybean trypsin inhibitor coupled to Sepharose 4B was capable of absorbing the IL-2 degrading activity from the trypsin solution. In contrast, alpha 2M.t treated with the solid-phase immobilized soybean trypsin inhibitor continued to inactivate IL-2, but did not degrade a macromolecular substrate (remazol-brilliant blue hide). Thus, IL-2 (MW 15,500) gains access to the active site of the alpha 2M-bound trypsin, resulting in a rapid loss of its biological activity. These observations offer an explanation for the in vitro immunosuppressive effects of alpha 2M.t.     

The immediate early genes of human cytomegalovirus upregulate expression of the interleukin-2 and interleukin-2 receptor genes.

Human cytomegalovirus (HCMV) immediate early (IE) genes act as trans-acting factors to upregulate various viral promoters. We used various IE plasmid constructs in transient transfection assays and demonstrated that the HCMV IE2 gene product upregulated expression from the interleukin (IL)-2 and IL-2 receptor (IL-2R) promoters and increased amounts of endogenous, steady-state IL-2 and IL-2R RNA. In marked contrast, the IE1 gene product, which can upregulate the major IE promoter and the IL-1 beta promoter, had no effect on the IL-2 and IL-2R promoters. These studies suggest a role for the HCMV IE2 gene product as a modulator of the inflammatory response associated with HCMV infection.     

An international study of human handedness: The data

Human handedness has been the subject of systematic study since 1646, but there is no agreement among researchers as to who can be considered a left-hander, what is the etiology of left-handedness, or what the proportion of left-handedness is in the world's population. This article reports the results of a handedness survey administered to 12,000 subjects in 17 countries, the largest handedness survey attemped. The paper discusses methods for determining handedness, the probability of a genetic component for handedness, and the relationship of sex, birth order, multiple birth, and first-degree relative's handedness on subject's handedness. A hypothesis for the etiology of left-handedness is presented.     

Concentrations of immunoreactive interleukin-1 and interleukin-2 in human preovulatory follicular fluid.

The presence of immunoreactive interleukin-1 (IL-1) and interleukin-2 (IL-2) in human follicular fluid obtained at the time of oocyte collection for in-vitro fertilization was ascertained by radioimmunoassay. In group I (20 fluids from 20 patients), the concentrations of IL-1 were 0.9 +/- 0.06 and 1.9 +/- 0.04 (mean +/- SEM) fmol/l in follicular fluid and plasma respectively. A positive correlation existed between IL-1 levels in follicular fluid and plasma (r = 0.56, P less than 0.01). Concentrations of IL-2 were 3.5 +/- 0.2 and 6.1 +/- 0.3 fmol/l in follicular fluid and plasma respectively. A positive correlation of IL-2 levels was also found between follicular fluid and plasma (r = 0.65, P less than 0.01). There was no association between IL-1, IL-2 and steroid levels, regardless of whether they were compared in follicular fluid or plasma. Group II was composed of a series of fluids (two to seven samples for each of seven patients) in which the follicular concentrations of IL-1 and IL-2 did not show a positive correlation with the volume of follicular fluid or the concentrations of follicular fluid steroids. It is concluded that human preovulatory follicular fluid contains immunoreactive IL-1 and IL-2. The role of IL-1 and IL-2 in ovarian physiology remains to be determined.