WHO Reference Preparation Research Articles

Long-Term Humoral Immune Response against SARS-CoV-2 after Natural Infection and Subsequent Vaccination According to WHO International Binding Antibody Units (BAU/mL).

The new WHO reference standard allows for the definition of serum antibodies against various SARS-CoV-2 antigens in terms of binding antibody units (BAU/mL) and thus to compare the results of different ELISA systems. In this study, the concentration of antibodies (ABs) against both the S- and the N-protein of SARS-CoV-2 as well as serum neutralization activity were evaluated in three patients after a mild course of COVID-19. Serum samples were collected frequently during a period of over one year. Furthermore, in two individuals, the effects of an additional vaccination with a mRNA vaccine containing the S1-RBD sequence on these antibodies were examined. After natural infection, the antibodies (IgA, IgG) against the S1-protein remained elevated above the established cut-off to positivity (S-IgA 60 BAU/mL and S-IgG 50 BAU/mL, respectively) for over a year in all patients, while this was not the case for ABs against the N-protein (cut-off N-IgG 40 BAU/mL, N-IgA 256 BAU/mL). Sera from all patients retained the ability to neutralize SARS-CoV-2 for more than a year. Vaccination resulted in a rapid boost of antibodies to S1-protein but, as expected, not to the N-protein. Most likely, the wide use of the WHO reference preparation will be very useful in determining the individual immune status of patients after an infection with SARS-CoV-2 or after vaccination.

A WHO Reference Reagent for lupus (anti-dsDNA) antibodies: international collaborative study to evaluate a candidate preparation

IntroductionAntibodies against double-stranded DNA (anti-dsDNA) are a specific biomarker for systemic lupus erythematosus (SLE). The first WHO International Standard (IS) for anti-dsDNA (established in 1985), which was used to assign...

Verification of the Roche cobas® 6800 PCR 200 µl and 500 µl protocols for the quantification of HIV-1 RNA, HBV DNA and HCV RNA and evaluation with COBAS® Ampliprep/COBAS® TaqMan® assays.

The analytical performance of the cobas 6800 HIV-1, HBV and HCV assays was verified and evaluated to the COBAS Ampliprep/COBAS TaqMan assays. The precision, limit of detection (LoD), limit of quantification (LoQ) and linearity were verified using pooled residual clinical samples. The analytical specificity was verified with negative plasma. HIV-1 analytical reactivity was verified with WHO reference preparations. Accuracy was verified using EQA plasma panels. Evaluation of the equipment was performed using prospectively collected clinical whole blood samples. Excellent precision was demonstrated using both testing protocols (coefficient of variation ≤15 %). The LoDs using the 500 and 200 µl protocols were 20 and 50 cp ml-1 for HIV-1, 10 and 20 IU ml-1 for HBV and 15 and 40 IU ml-1 for HCV, respectively. The LoQs were 40 and 100 cp ml-1 for HIV-1, 20 and 25 IU ml-1 for HBV and 30 and 80 IU ml-1 for HCV, respectively. Assays demonstrated good linearity (R2 ≥0.96). The analytical specificity of the assays was 100 %. There was excellent agreement between the cobas 6800 and CAP/CTM assays (kappa>0.94). The sensitivity, specificity, positive predictive value and negative predictive value for each of the assays were ≥99 %. The cobas HIV-1 and HCV mean quantifications were 0.03 log10 cp ml-1 and 0.17 log10 IU ml-1 higher than the CAP/CTM. The cobas HBV mean quantification was 0.17 log10 IU ml-1 lower than the CAP/CTM. Subtype/genotype specific differences were not observed. Cobas 6800 equipment and assays demonstrated excellent performance and correlated well with CAP/CTM assay results.

EUVAC.NET collaborative study: Evaluation and standardisation of serology for diagnosis of pertussis

As part of the new EUVAC.NET contract with ECDC (Pertussis Work Area 4), a collaborative study was organised in July–December 2010. Two well-defined reference preparations with high and low IgG antibodies to pertussis toxin (PT), were sent to participants. The purposes of this study were to assess current laboratory performance of serological assays for pertussis; to compare in-house reference preparations that are currently used by participants for the serological assay; and to identify needs for standardisation of the serological assay. Reference Laboratories in Europe currently performing serological assays for the diagnosis of pertussis by measuring antibody to PT, were invited to participate in the study. A total of 17 laboratories/countries participated in this study. Results were reported from a total of 9 participants who used in-house ELISA assays and 10 participants who used commercial kits. All participants using in-house ELISA with purified PT coating plates distinguished the 2 preparations and gave results that were comparable to the expected values. A total of 6 commercial kits included in the study showed different results. The kits coated with mixture antigens did not appear to be able to give results that were correlated to the WHO reference preparations.

Optimisation of sandwich ELISA based on monoclonal antibodies for the specific measurement of pregnancy-associated plasma protein (PAPP-A) in acute coronary syndrome

Objectives: PAPP-A has become the principal biochemical serum marker in first trimester screening for Down syndrome, the original data being based on results of a radioimmunoassay (RIA). Recent observations using sandwich ELISA technology have proposed PAPP-A as a potential marker in patients with acute coronary syndrome (ACS). The aims of the present study were to demonstrate (i) the importance of antibody specificity, (ii) the potential pitfalls in changing assay technology, (iii) the importance of strict definition of technology, and (iv) the application of a well-defined assay technology on sera from patients with ACS. Design and methods: Candidate monoclonal antibodies (Mab) were identified by immunohistochemistry, Western blot and the absence of positive signals (ELISA) with normal, non-pregnant serum as antigen source. The ELISA technology was standardized against the original PAPP-A RIA and the WHO reference preparation (WHO 78/610). Results different from those obtained by the original RIA led to ELISA modifications with respect to dilution buffer and enzymatic digestion of the Mab. Results: The first generation ELISA revealed serum measurements from a pool of non-pregnant ( n = 103) individuals which, compared to the RIA, seemed to be false positive. The false positive reaction was abolished by addition of bovine serum (BS) to the dilution buffer. Subsequent analysis of individual sera ( n = 103) indicated that 7/103 were still false positive. This reaction was eliminated by introduction of F(ab′) 2-fragment of the indicator antibody. This modified ELISA revealed that serum PAPP-A levels in ACS were statistically significantly higher than in controls ( p < 0.001). Moreover, serum PAPP-A in ACS patients with ST-segment elevation (STEMI) were higher ( p < 0.001) compared to patients without ST-segment elevation (NSTEMI). Immunohistochemical analysis failed to identify PAPP-A in the atherosclerotic plaques.

Quantitative Measurements of Allergen Specific IgG4 and IgG Antibodies using ImmunoCAP Technology

RATIONALE: Analytical performance of ImmunoCAP technology for quantitative measurements of allergen specific IgG4 and IgG antibodies was evaluated. METHODS: Parallelism of the IgG and IgG4 calibrators to WHO reference preparation (WHO 67/86) was evaluated. The cross-reactivity of the anti-IgG4 and anti IgG conjugates to other immunoglobulines and different subclasses of IgG were tested and precision of each test was evaluated. ImmunoCAP systems provided automated handling with short assay times (less than 3 hours). Samples were assayed at a standard dilution of 1:100. The analytical performance of the new test was further verified by testing the IgG4 and IgG antibody levels to cat and dust mite allergens in 738 child samples and 100 adult blood donors. RESULTS: Stepwise dilutions of samples were parallel to the calibrator curve assuring quantitative measurements of IgG4 and IgG antibodies. Cross-reactivity was < 0.5% Precision (total CV%) was below 15% over the measuring range. Median values for IgG4 antibody concentrations to mite extract(d1) were 0.23 and 0.25 mgA/l in the healthy donors and child samples respectively and the corresponding values for cat extract were 0.45 and 0.50 mgA/l. CONCLUSIONS: ImmunoCAP systems are reliable tools for quantitative measurements of allergen Specific IgG4 and IgG antibodies.

Isolation of hCG and its Characterization by Radioimmunoassay, Enzyme‐Immunoassay, and Radio‐Receptor Assay

The nature of human chorionic gonadotropin (hCG) molecules present during early pregnancy of Indian women is poorly understood. Therefore, a study has been undertaken to isolate hCG and characterize different forms of hCG from urine. The hCG molecules from urine of pregnant women (45–75 days post LMP) were adsorbed onto kaolin, eluted with ammonium hydroxide, and precipitated using acetone and then lyophilized. The lyophilized extract was subjected to Sephadex G‐100 chromatography followed by ion‐exchange fractionation. Three major fractions of protein (i.e., Peaks I, II, and III) associated with carbohydrate activity were obtained. Peaks II and III eventually resolved into a single peak I following repeated ion exchange chromatography, which suggested the presence of aggregates of molecules. Further purification on an affinity column resolved all three peak fractions into one unadsorbed and two adsorbed (A and B) fractions. These adsorbed fractions were characterized by radioreceptor assay (RRA), radioimmunoassay (RIA), and enzyme linked immunosorbent assay (ELISA). The activity was standardized against WHO reference preparation 75/589. Peaks I (A and B) were found to have maximum at about 75% of immunologically potent hCG, followed by peaks II (40%) and III (5%). The molecular sizes of peaks I, II, and III on a Sephadex G‐200 column corresponded to 27,500D, 66,000D, and 84,000D, respectively. Relative mobilities of all adsorbed fractions in sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) showed the presence of hCG‐α (mol. wt. 19,539D) and hCG‐β (28,870D) subunits. The presence of both subunits of hCG were also revealed by Western blot analysis. For the first time, we report the low molecular weight hCG molecule, of 27,500D by size exclusion chromatography, which has immunological and biological activity as measured by RIA, ELISA, and RRA.

THE TURBO-MP RADIOIMMUNOASSAY FOR MEASUREMENT OF FOOD-SPECIFIC IMMUNOGLOBULIN E. - FP1

Background: The accurate measurement of food specific IgE is considered an important parameter in determining the etiology of adverse reactions to foods. The Turbo-MP test has recently been developed, featuring multipoint calibration based on the WHO reference preparation and providing over 200 food allergens, out of a total of over 1000 allergens available. Objective: The purpose of this investigation was to determine the inter-assay and inter-laboratory reproducibility of the Turbo-MP assay, and to investigate the correlation of the quantitative results of Turbo-MP with those of the FEIA methodology for food-specific IgE. Methods:A retrospective study was performed on serum samples selected from a clinical reference laboratory on the basis of specificities for major food allergens. Results: Correlation of quantitative results was determined for two successive assays performed using samples with specificities for five foods (egg white, cow’s milk, codfish, peanut, soybean), resulting in a slope 1.04 (95% confidence interval 1.00-1.07) and Pearson’s coefficient 0.99. Correlation of quantitative results from two laboratories performing theTurbo-MP assay on the same samples showed a slope of 0.88 and Pearson’s coefficient of 0.97. A plot of RIA vs. FEIA test results greater than 0.35 IU/mL (n = 343) showed a correlation with a Pearson’s coefficient of 0.68 (95% confidence interval 0.62 to 0.73); slope of 0.94 (95% confidence interval 0.84-1.0); and intercept of -0.04 (95% confidence interval -0.240.27).A cut-off of 0.35 IU/mL was selected due to the inherent limitation in the FEIA method.The data are distributed symmetrically around the equivalence line (x=y), and variations seem to be due primarily to differences in the allergens used in the solid phase.The decision points proposed by Sampson et al. (JAllergy Clin Immunol 1997; 100(4): 444-451) were used to score results from both tests in order to compare how each test would predict reactivity to specific foods. The overall concordance of the Turbo-MPwithFEIAwas80.5%(eggwhite84.6%;cow’smilk79.2%;codfish75.8%; wheat 80.3%. peanut 90.6%; soybean 76.4%; tuna 73.2%). Conclusions:We conclude that theTurbo-MP assay correlates well with the FEIA method, is valuable in the accurate quantitation of food specific IgE, and can be used in diagnostic schemes such as that proposed by Sampson et al. FP2 Identification of Proteins Differentially Expressed in Sera of Patients with Atopic Dermatitis by Proteome Analysis.

A sensitive non-isotopic assay for GAD 65 autoantibodies

Background: A new ELISA for 65 kDa isoform of glutamic acid decarboxylase autoantibodies (GAD 65 Abs), which depends on GAD 65 Ab acting divalently and forming a bridge between immobilized GAD 65 and liquid-phase GAD 65–biotin, is described. Methods: Sera (25 μl) were incubated in GAD 65-coated ELISA plate wells followed by washing and incubation with GAD 65–biotin. After a further wash step, GAD 65–biotin bound was quantitated by addition of streptavidin peroxidase followed by tetramethylbenzidine. Assay calibration was with WHO reference preparation 97/550. Results: Using a cut-off for positivity of 5 units/ml, sera from 0.7% (2/300) healthy blood donors (HBDs), 100% (39/39) selected type 1 diabetes mellitus (DM) patients, 1.6% (1/62) type 2 diabetes mellitus patients and 3% (4/119) autoimmune disease controls were GAD 65 Ab positive in the ELISA. Levels of positivity in an immunoprecipitation assay (IPA) based on 125I-labelled GAD 65 (cut-off 25 units/ml) were 1%, 82%, 0% and 3%, respectively. ELISA inter-assay coefficients of variation ( n=12) were 7.3%, 3.8%, 7.2% and 6.3% at 5.4, 40.8, 137 and 382 units/ml, respectively. Conclusions: The ELISA we have described has sensitivity and specificity at least as high as current radioactive assays. It has good precision and handling making it suitable for routine use.

A Precise and Rapid Microtitre Plate Clot Lysis Assay: Methodology, Kinetic Modeling and Measurement of Catalytic Constants for Plasminogen Activation during Fibrinolysis

A rapid and precise turbidimetric clot lysis assay employing a microtitre plate reader and personal computer is described in detail. The use of such widely available instrumentation, the convenience and rapid throughput suggest the assay could be developed as a reference method with which to measure the potency of tissue plasminogen activator (t-PA) in conjunction with the WHO reference preparation. The method has been used to investigate molecular parameters involved in fibrinolysis. Aggregation status of the fibrin does not appear to influence the mechanism of plasminogen activation and clot lysis by plasmin. High ratios of plasminogen to fibrin resulted in a change in clot turbidity and in a change in the lysis profile of turbidity versus time. This is probably the result of plasminogen binding to fibrin and consequent restriction of the access of plasmin to its sites of cleavage in the fibrin. A simple model is proposed, and equations have been derived, for the kinetics of lysis which adequately describe the mechanism and which are confirmed by experimental data. This model results in estimates of the Km and kcat for the activation of plasminogen by t-PA during clot lysis of approximately 150 nM and 0.1 s-1, respectively, in excellent agreement with published values. The assay should therefore prove useful in quantitative evaluations of the molecular phenomena occurring during fibrinolysis. The more rapid activation of lys-plasminogen than glu-plasminogen by t-PA was confirmed. However, evidence was obtained that the lys-form binds more tightly to fibrin by the same factor. This observation suggested that the appropriate substrate in the kinetic model is fibrin-bound plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Standardisation of the quantitative determination of antinuclear antibodies (ANAs) with a homogeneous pattern.

The results of antinuclear antibody tests using the indirect immunofluorescence technique may be reported as a description of the pattern and the intensity of fluorescence obtained at a certain dilution. If quantitative results are required titration is necessary. Such titrations may vary greatly between different laboratories. The present study involving 26 laboratories shows an improvement of interlaboratory comparability for the homogeneous fluorescence pattern when a common reference serum is used. Cultured cells as substrate appear to give better quantitative agreement than rat liver sections. National reference sera should be standardised in items of the appropriate WHO reference preparation.

Quantitation of IgM, IgA and IgG rheumatoid factors by ELISA in rheumatoid arthritis and other rheumatic disorders.

We describe the evaluation of an enzyme-linked immunosorbent-assay (ELISA) for the measurement of IgM, IgA and IgG rheumatoid factor (RF). Results were obtained from sera of normal controls and patients with different rheumatic disorders in a cross-sectional study. Both human and rabbit IgG could be used as antigen in the assays. IgA-RF and IgG-RF were measured after pepsin digestion. The WHO reference preparation was used to standardise IgM-RF, but could not be used in the IgA- and IgG-RF assay, because neither IgA- nor IgG-RF could be detected in this standard serum. The ELISA for detection of RF is a highly sensitive and reproducible method and quantitation of results can be standardised. It yields good discriminative values for RA versus controls, in particular when more RF-isotypes are determined. In RA patients a correlation between IgM-, IgA- and IgG-RF concentrations does exist, although a wide variation was found in the ratio of these concentrations.

An international collaborative study on standardization of apolipoproteins A-I and B. Part I. Evaluation of a lyophilized candidate reference and calibration material.

We evaluated a lyophilized serum preparation for use as a candidate Reference Material for apolipoproteins (apo) A-I and B. An international collaborative study was conducted with 28 participating laboratories, selected on the basis of participation and demonstrated expertise in a 1983 survey of apolipoproteins A-I and B. The analytical suitability of the material was established by confirming linearity of its dose-response curves over a desired concentration range and demonstrating that its response curves paralleled those for fresh sera. Differences in dilution-adjusted mass units ascribable to the five analytical methods used by the various laboratories constituted only 1% of the total variation for apo A-I, but 32% for apo B. The dominant source of error, however, for both apo A-I and B was the variability among laboratories, rather than variability among methods and antisera. The assigned consensus mass-concentration units based on study data are 1.124 g/L for apo A-I, 0.589 g/L for apo B. For these estimates the coefficients of variation were 13% and 27%, respectively. These findings on the proposed Reference Material meet the requirements suggested by the World Health Organization's Expert Committee on Biological Standards for a candidate WHO Reference Preparation.

Intra-individual variation in the concentrations of IgA, IgG, IgM, and complement component C3 in serum of a normal adult population.

A recent study [Clin. Chem. 22, 1635 (1976)] reported intra-individual variation in 10 serum proteins to be much smaller than interindividual variation. We report results of a similar study involving about 700 apparently healthy adults in whom we estimated the relative magnitude of the intra- and interindividual variation in serum IgA, IgG, IgM and complement component C3. Specimens were collected from each subject weekly for as long as 10 weeks (average, four weeks). The four serum proteins were quantitated by radial immunodiffusion by the maximal-diffusion technique. Traditional 95% reference intervals were computed relative to WHO reference preparations for the immunoglobulins. For C3, the reference interval was computed relative to a commercial reference preparation. We, too, found the ratios of intra-individual to interindividual variation for adults to be so small that the traditional reference intervals do not have the assumed diagnostic sensitivities. Furthermore, these ratios did not change after dividing the study population into subgroups according to sex and age; evidently such subgrouping do not improve the diagnostic sensitivity. The relatively small intra-individual variations were also observed at the extremes of protein concentration ranges.

The validity of the radial immunodiffusion method for the quantitative determination of human IgM. Evaluation of a modified method

Three subgroups of human IgM can be distinguished on the basis of differences of slopes of the D 2 versus absolute concentration plot in the radial immunodiffusion technique (Klein et al., 1973). Individual IgM fractions, whether mono- or polyclonal, always belong to only one of these groups. The differences between the subgroups disappear after reduction of the IgM to 7S subunits. These findings provide an explanation for the large discrepancies between absolute IgM determinations in different laboratories. It follows that most of the readings of individual IgM values in the Mancini technique must be wrong by any standard, including WHO reference preparations. The insertion of a simple reduction step in the assay abolishes the differences in quantitative reactivity between IgM subgroups as well as between natural 7S and 19S IgM. This allows an absolute determination of both forms together. The values thus obtained differ considerably from the estimates given by Humphrey and Batty (1974). It also appears that the International Units of the WHO do not represent the same quantity of IgM in different reference sera. The modified method allows the determination of total monoclonal as well as polyclonal human IgM by weight.