Developments in immunological standardization

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Abstract

In the century since Paul Erlich's innovative immunological standarization work with diphtheria anti-toxin, the field of immunological standardization has expanded dramatically. Biological standards for a diverse range of immunological substances have been produced e.g. immunoglobulins, complement components, autoantibodies and blood group reagents. The concept of calibration of such materials in biological units of potency is now widely accepted for many such substances. Most recently much effort has been devoted to producing biological standards for cytokines which can be used to calibrate and validate biological assays for these analytes. Immunoassays have been found to be particularly problematical from the standardization view point although provision of a single international standard for distribution world-wide is clearly advantageous and helps reduce assay variability. It is hoped that the considerable progress with immunological standardization achieved during the past century will continue and expand to ensure the validity of existing and new immunological assays which will be required in the future.

Introduction

The 25th birthday of the Journal of Immunological Methods virtually coincides with the 100th anniversary of Paul Ehrlich's pioneering work in the biological standardization field. In 1897, he established a biological unit for diphtheria antitoxin which was defined as a portion of the content of a particular `standard' preparation of dried antitoxin. Prior to this the potency of antitoxin preparations (and many other biologicals) had been determined by measuring some assay parameter, such as a 50% maximal response, or the ability to neutralise a stated quantity of toxin which had clearly shown dramatic variability and inaccuracy in most if not all assay systems. This lack of comparability and variation in results obtained from assays was largely due to differences in the determination and expression of results and differences in responses obtained in vivo in different species. The expression of results using a plethora of different unitage systems (including the notoriously incomparable in vivo derived LD50 mouse, rat, cat, frog, etc. units) compounded problems of consistency and comparability of assays for a wide range of biological substances. Ehrlich's laboratory-based investigations clearly indicated that use of a single standard preparation world-wide for calibration of assays allowed direct comparison of results obtained in different laboratories at different times and produced using a wide range of assay formats. This solved many of the problems encountered previously when no standard was used for calibration purposes. The `Ehrlich' standard for diphtheria toxin was distributed widely and despite problems encountered during World War I, its unitage was maintained by the use of similarly calibrated secondary standards. In 1922 the League of Nations adopted the Ehrlich unit as the International unit, instigating the concept of a world-wide status for standard preparations and their associated unitage. This activity has been maintained (later by WHO of the United Nations, which succeeded the League of Nations) to the present.

Subsequent to this many other International Standards were produced for a wide range of biologicals and Madsen, Dale and Hartley can be credited for ensuring that such efforts were pursued with vigour and appropriate scientific thoroughness. The Expert Committee on Biological Standardization (ECBS) of WHO has overseen developments since the World War II and establishes appropriately characterized preparations of biological materials as WHO International Standards, Reference Preparations or Reference Reagents. By 1974 there were more than 270 of these standard preparations for antigens, antibodies, antibiotics, vitamins, hormones and enzymes.

Standards and Reference Preparations/Reagents for immunological materials were early candidates for establishment by WHO (with international status) and other organizations. Amongst a list of biological reference materials published in 1971 and distributed from the Division of Biological Standards, National Institute for Medical Research, UK were standards for various autoimmune sera, immunoglobulins G, A, M, D and E, rheumatoid factor and interferons. (Anon, 1971).

Section snippets

Uses of standards

Standards are primarily intended for the calibration of assays, although they are also valuable for assay validation. In some cases they can also be used for comparing the performance of different assays and their ability to discriminate/detect different or similar analytes. As Ehrlich demonstrated, the most important characteristic of a standard is its associated unitage, which is defined by the analyte content of the ampoule of standard. Assays are calibrated in terms of the dose–response

Unitage of standards

Ehrlich, Dale and others showed that calibration of standards in units of activity which relate to the ampoule contents was essential if their use was to be universal and valid. Calibration in terms of an assay response always caused comparability and other problems (for an early amusing and still valid discussion of this see Burn, 1930). Subsequent studies have shown that calibration of biological standards in terms of `mass' or `weight' is not possible if performance in all assays with all

Production and availability of standards

Processes for producing, evaluating, establishing and distributing International Standards were devised many years ago by WHO. These have stood the test of time and still remain in place (World Health Organisation, 1990). Non-WHO Standards/Reference Preparations or Reference Reagents are often not produced using the same criteria and in some cases this adversely affects their usefulness and validity, at least for calibration purposes.

Initially, candidate materials made available for

Developments in standardization over the past 25 years

A general expansion in standardization activities has occurred over the past 25 years, as the number of analytes requiring standards has increased but the general principles followed have not substantially changed. However, International Standards for some small molecules, such as vitamins and some antibiotics have been discontinued as these materials can be measured using physiochemical procedures.

In the immunology field the most significant group of molecules for which standards have been

Future trends in standardization

Standards for biological analytes will clearly continue to be needed for the foreseeable future. The production of variant and mutant proteins using rDNA technology which have different biological potency/activity to the parent (naturally occurring) substance, may require the generation of additional reference preparations for such molecules.

The expansion in the use of methods based on the polymerase chain reaction (PCR) to detect mRNA for a range of immunologically important molecules

Problems with standardization

Despite active progress with immunological standardization over the past 25 (if not 100!) years, problems with standardization of immunological (and other) assays remain. These generally compromise the validity and accuracy of results obtained and also cause lack of comparability of data derived in different experiments at different times and especially in different laboratories. The most obvious problems arise when no standard preparation is used at all, i.e. the same problems as originally

Conclusions

Most procedures used in immunological investigations are indirect methods in which samples with an unknown content of analyte are assayed by measuring some assay-related parameter, such as absorbance of light at a defined wavelength, cpm produced by a particular radioisotope, etc. The only valid approach for calibration of such procedures is by comparison with a standard preparation with defined analyte content. It is therefore crucial that the standard used is appropriate and available

Acknowledgements

I am grateful to my colleagues Chris Bird, Tony Mire-Sluis and Meenu Wadhwa for discussions relating to this paper and to Jenni Haynes for preparing the manuscript.

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