R.E. Gaines Das

Development of radioimmunoassays for human erythropoietin using recombinant erythropoietin as tracer and immunogen.

Since the estimation of erythropoietin (EPO) by radioimmunoassay (RIA) has been limited by the availability of highly purified urinary (U) human (Hu) EPO, we investigated the use of recombinant (R)-HuEPO as a replacement for U-HuEPO in the preparation of 125I-tracer and high affinity antisera. In each of two validated RIAs developed using U-HuEPO-derived reagents, dose-response lines and potency estimates for samples were compared when 125I-U-HuEPO and anti-U-HuEPO antisera were sequentially replaced to give assay variants using R-HuEPO-derived reagents. Two U-HuEPO preparations, the 2nd International Reference Preparation and CAT-1, and R-HuEPO were variously used as standards. The samples tested included clinically relevant human sera and partially purified preparations of U-HuEPO and R-HuEPO. In each RIA and for each assay variant tested, samples gave dose-response lines whose slopes did not differ significantly from that of the standard. For each of the two variant RIAs, potency estimates for any sample were consistent and, where examined, RIA potency estimates agreed with in vivo bioassay determinations. These results, obtained independently in two laboratories, indicate that RIAs having appropriate specificity and sensitivity for the estimation of EPO in clinical samples can be developed using reagents derived from R-HuEPO.

The World Health Organization International Collaborative Study for Islet Cell Antibodies

Aims/hypothesis. Islet cell autoantibodies are a specific marker for Type 1 (insulin-dependent) diabetes mellitus. Standardisation of islet cell antibodies and the uniform reporting in International units is critical to research and the development of assays for islet cell autoantibodies as diagnostics.¶Methods. The suitability of a candidate serum to serve as the international standard for islet cell antibodies was studied by 19 participants in 8 countries. In addition, the purpose was to investigate whether the serum could also serve as a standard for antibodies to the 65 000 Mr isoform of glutamic acid decarboxylase (GAD65) and islet antigen-2 (IA-2). Control sera were included in the study to assess the validity of the various assay systems. The sera were lyophilized to World Health Organization criteria and the candidate serum assigned the ampoule code number 97/550.¶Results. The use of 97/550 was shown to notably reduce inter laboratory variability in the measurement of islet cell antibodies. In addition, there was a pronounced reduction in inter laboratory variability in the measurement of GAD65 and IA-2 antibodies. ¶Conclusions/interpretation. On the basis of the results reported here and with agreement of the participants, the preparation 97/550 has been established by the World Health Organization Expert Committee on Biological Standards for establishment as the first international standard for islet cell antibodies, with an assigned potency of 20 international units. In addition, 97/550 can serve as an international reference reagent for specific GAD65 antibodies, with an assigned potency of 100 units. It can also serve as a National Institute of Biological Standards and Control (NIBSC) reference reagent for IA-2 antibodies for evaluation of assays for this material. [Diabetologia (2000) 43: 1282–1292]

Peripheral analgesic activities of peptides related to α‐melanocyte stimulating hormone and interleukin‐1β193–195

1 The hyperalgesic effects of interleukin‐1β (IL‐1β) and prostaglandin E2 (PGE2) were measured in rats. 2 Hyperalgesic responses to IL‐1β were inhibited in a dose‐dependent manner by α‐melanocyte stimulating hormone (α‐MSH)‐related peptides with the following order of potency: [N14,d‐Phe7]α‐MSH > α‐MSH > Lys‐d‐Pro‐Val > Lys‐Pro‐Val > Lys‐d‐Pro‐Thr > d‐Lys‐Pro‐Thr. 3 Hyperalgesic responses to PGE2 were not inhibited by Lys‐d‐Pro‐Thr and d‐Lys‐Pro‐Thr but were inhibited in a dose‐dependent manner by the other peptides with the same order of potency as against IL‐1β. 4 The potencies of [N14, d‐Phe7]α‐MSH and α‐MSH were greatly diminished by deletion of their C‐terminal tripeptide, Lys11‐Pro‐Val13. 5 Nor‐binaltorphimine (Nor‐BNI) largely reversed the analgesic effects of α‐MSH, [N14, d‐Phe7]α‐MSH, Lys‐Pro‐Val and Lys‐d‐Pro‐Val indicating that κ‐opioid receptors mediated the analgesic activity of these peptides. 6 Nor‐BNI did not antagonize the inhibition by Lys‐d‐Pro‐Thr and d‐Lys‐Pro‐Thr of IL‐1β evoked hyperalgesia indicating that these peptides were not acting via κ‐opioid receptors.

Implications of experimental technique for analysis and interpretation of data from animal experiments: outliers and increased variability resulting from failure of intraperitoneal injection procedures

Intraperitoneal (i.p.) injections are widely used in laboratory animal experiments. This technique has a failure rate that is typically reported to be of the order of 10–20%. It is not apparent that failures of i.p. injection and their consequences for the experimental results are as widely recognized as the use of the technique. We illustrate the consequences of i.p. injection failure for the analysis and interpretation of several bioassays. We suggest approaches to data analysis that should be considered, and emphasize the need to recognize and allow for the possibility of i.p. injection failure in the analysis and interpretation of laboratory animal experiments involving this technique.

Cytokine levels as performance indicators for white blood cell reduction of platelet concentrates.

Background and Objectives With the implementation of universal white blood cell (WBC) reduction in the UK, in‐process WBC‐reduction filters for pooled buffy coat (BC)‐derived platelet concentrates (PCs) and apheresis methods are used routinely for the production of WBC‐reduced PCs. While these strategies meet the specification for WBC reduction (< 5 × 106 WBCs/unit), the products from these processes may differ depending on the process employed and its performance. The aim of this study was therefore to investigate whether PCs prepared using various WBC‐reduction processes are sufficiently depleted of WBCs to limit cytokine accumulation during storage and to assess if cytokine levels detected in platelet products can serve as indicators of acceptable platelet activation as a result of the WBC‐reduction process.

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.

Second international standard for endotoxin: calibration in an international collaborative study

On behalf of the World Health Organization, 26 scientists in 13 countries calibrated a candidate international standard for endotoxin and compared it with the Pharmacopoeial standards of the US, Europe and Japan. The study showed that the candidate standard is suitable to serve as the second international standard for endotoxin for all applications, with an assigned unitage of 10,000 international units (IU)/vial, with 1 IU = 1 EU (FDA/USP endotoxin unit). This value maintains continuity of unitage with EC5 (the primary calibrant for the first international standard), EC6 (the current FDA/USP standard), the Japanese Pharmacopoeia Standard and commercial preparations of Limulus lysate. This value is also consistent with calibration of the candidate standard in terms of the European Pharmacopoeia Standard, BRP-2. The establishment of this international standard for endotoxin will permit the global harmonisation of unitage for standards of endotoxin.

WHO international reference reagents for human proinsulin and human insulin C-peptide.

Candidate preparations for international reference reagents for immunoassays of human proinsulin and human insulin C-peptide were evaluated in an international collaborative study. With the authorization of the Expert Committee on Biological Standardization of WHO, the following preparations were established as international reference reagents: human proinsulin (84/611, nominal ampoule content 6 micrograms) and human insulin C-peptide (84/510, 10 micrograms). Each preparation is intended as a primary reference reagent for the calibration of immunoassays.

Comparison of the bioactivity of reference preparations for assaying Bordetella pertussis toxin activity in vaccines by the histamine sensitisation and Chinese hamster ovary-cell tests: assessment of validity of expression of activity in terms of protein concentration.

Pertussis toxin (PT) in its detoxified form is an important antigenic component of both acellular and whole cell pertussis vaccines. Limits on the content of active PT in acellular vaccines are set in official monographs (EP, WHO, USP) and evidence of compliance is therefore, required by regulatory authorities. The two assay methods which are currently used by most manufacturers and official national control laboratories to monitor residual PT activity in acellular pertussis vaccines (and also in whole cell vaccines) are histamine sensitising (HIST) assays and Chinese hamster ovary (CHO) cell assays. Currently, different reference preparations of PT are used by individual laboratories for these tests. We therefore organised an international collaborative study to examine, by these two assay methods, two freeze-dried purified preparations of PT, one preparation in ampoules coded JNIH-5 and one preparation in ampoules coded 90/518, together with in-house reference (IHR) preparations in current use. Data from this study confirm that both JNIH-5 and 90/518 show biological activity both in HIST assays and in CHO-cell assays. Both HSD50 and ED50 values obtained in this study differ significantly between laboratories and thus show that biological activity is not determined by the nominal masses of preparations. Estimates of relative potency of 90/518 in terms of JNIH-5 per ampoule for the HIST assays do not differ significantly between laboratories. The overall mean estimates of relative potency of 90/518 in terms of JNIH-5 do not differ significantly between the two methods. Data from this study further indicate that the biological activity of different preparations was not directly related to their stated protein content. The use of protein content to indicate the level of PT activity in different preparations would give misleading results. Thus, use of a common standard is shown to greatly improve between laboratory agreement of estimates.

Serum immunoreactive erythropoietin in patients with idiopathic aplastic and Fanconi's anaemias

Summary. In patients with idiopathic aplastic anaemia (n = 34) and Fanconis anaemia (n = 8), sampled once or on several occasions, serum erythropoietin (Epo) increased with increasing severity of anaemia with apparently similar rates of increase in each group. However, after adjustment for Hb, log Epo values for the Fanconis anaemics tended to be greater than those for the idiopathic aplastic anaemics (P < 0·01).