A New Eu³⁺‐Labeled Method for Anticardiolipin Antibody IgM

Abstract

Background

The anticardiolipin antibodies (aCL) test has become a laboratory standard for the clinical diagnosis of antiphospholipid syndrome (APS). To better the quantitative detection of aCL‐IgM so as to classify patients correctly and timely as APS positive, we established herein a new immunoassay based on a time‐resolved fluoroimmunoassay (TRFIA).

Methods

The complex of cardiolipin plus bovine anti‐β₂ glycoprotein‐I was used as antigen fixed on microtiter plates to detect serum aCL‐IgM, and Eu³⁺‐labeled rabbit antihuman IgM was used as conjugate. The precision, sensitivity, specificity, coefficient of recovery, and stability of the assay were evaluated, and comparison with the traditional, classical enzyme‐linked immunosorbent assay (ELISA) was also made.

Results

The detection limit of the aCL‐IgM TRFIA kit we established was 0.1 MPL U/ml, with a wider detectable range than commercial ELISA ones when a strong‐positive specimen was diluted from 2,630.9 to 0.08 MPL U/ml. There was a good liner range within 0.16 to 2,630.9 MPL U/ml, whereas it was within 5.14 to 328.86 MPL U/ml when using three commercial ELISA ones. The average intra‐ and interassay variability was 3.19 and 3.70%, respectively. The mean recovery rate was 101.95%. The clinical diagnostic specificity was 98%. Additionally, the established assay kit presented good characteristics of stability and correlated well with the ELISA, and the correlation coefficient was 0.955.

Conclusion

The aCL‐IgM TRFIA provides an approach to a more sensitive and reliable diagnosis of APS. Further validation of its use is required.

INTRODUCTION

The anticardiolipin antibodies (aCL) test, first reported by Harris in 1983, has been proved to be predictive in identifying patients with antiphospholipid syndrome (APS) that subjects people to recurrent arterial and/or venous thrombosis and recurrent pregnancy losses, in the presence of, as in most cases, the persistently increased titer of antiphospholipid antibodies (aPL) as serologic markers (some patients do have clinical signs suggestive of APS but persistently test negative for aPL) 1, 2, 3. Since APS patients can behave very differently with a variety of clinical symptoms, it is quite a challenge for clinicians to classify them correctly as APS positive. Hence, appropriate laboratory testing is extremely necessary to assist the clinical diagnosis of the disease. The revised laboratory APS criteria updated in 2006 recommended that patients clinically suspected for APS should be excluded or confirmed by coagulation tests in vitro for the detection of the lupus anticoagulant (LA), solid‐phase ELISA for measuring aCL and/or β₂GPI (glycoprotein‐I; 2). As the representative pathogenic aPL, aCL antibodies of elevated level have crucial implications for clinical and therapeutic management of APS 4, 5. The assay of ELISA, the dominant assay currently used for aCL detection, however, still shows methodological and diagnostic shortcomings, particularly, its low sensitivity and poor measurement accuracy, which cripple the screen role of the test. With respect to the different isotypes of aCL, aCL IgG and aCL IgM are generally considered to be more strongly associated with the clinical manifestation of APS than aCL IgA 6, 7, 8 and the detection of aCL IgM antibodies theoretically reflects an early stage of the disease opposed to aCL IgG that reflects a sustained class‐switched immune response. We herein develop a new immunoassay for aCL IgM detection based on time‐resolved fluoroimmunoassay (TRFIA), which has been recently proposed as a more sensitive alternative to classical ELISA 9, 10. The complex of cardiolipin plus bovine β₂GPI was used as antigen and Eu³⁺‐labeled rabbit antihuman IgM as conjugate. The new established assay demonstrated a higher diagnostic sensitivity, wider detectable range as well as better stability than the traditional aCL ELISA.

MATERIALS AND METHODS

Chemicals and Instruments

Solid‐phase antigen (the complex of cardiolipin from bovine heart plus bovine β₂GPI), diethylenetriamine tetraacetic acid (DTPAA), and monoclonal rabbit antihuman IgM were purchased from Sigma (St. Louis, MO). Ninety‐six‐well polystyrene microtiter plates were obtained from Nunc International (Roskilde, Denmark). Bovine serum albumin (BSA) was from the Department of Health, Institute of Biological Products (Shanghai, China). Eu³⁺‐labeling kit was from PE company (EG&G Wallac, Finland). PD‐10 column and sepharose CL‐6B column (1 × 40 cm) were from the Pharmacia company (Piscataway, NJ). Three commercial ELISA kits for aCL‐IgM detection were purchased from Orgentec Diagnostika Gmbh (Naina, Germany), Dr. Fenning BioMed GmbH (Kirchzarten, Germany), and AESKU (Wendelsheim, Germany), respectively. Auto DELFIA₁₂₃₅ TRFIA analyzer was from Perkin‐Elmer Life and Analytical Science/Wallac Oy (Turku, Finland). All additional chemicals and reagents used were of analytical grade.

Reagent Solutions

Labeling buffer contained 50 mmol/l Na₂CO₃–NaHCO₃ (pH 8.5) and 0.155 mol/l NaCl. Elution buffer contained 50 mmol/l Tris‐HC1 (pH 7.8), 0.9% NaC1, and 0.05% NaN_3. Assay buffer contained 50 mmol/l Tris‐HCl, pH 7.8, containing 0.9% NaCl, 0.2% of purified BSA, 0.01% Tween 20, 20 μmol/l DTPA, and 0.05% sodium azide. Washing solution was a Tris‐HCl buffered saline solution (pH 7.8) containing 0.9% NaCl, 0.2% Tween 20, and 0.05% NaN₃. Enhancement solution was a 0.1 mol/l acetate–phthalate buffer (pH 3.2) containing 0.1% Triton X 100, 15 μmol/l βNTA, and 50 μmol/l tri‐n‐octylphosphine oxide.

Solid‐Phase Antigen Preparation

The complex of cardiolipin from bovine heart plus bovine β₂GPI was diluted with 50 mmol/l Na₂CO₃–NaHCO₃ buffer (pH 9.6), and then 200 μl was added to each well of 96‐well polystyrene microtiter plates. After being incubated overnight at 4°C, the coating buffer was discarded and each well was blocked with 250 μl 3 g/l BSA (dissolved in PBS, pH 7.4) and then incubated at a room temperature for 2 h in a miniorbital shaker. The blocking buffer was discarded and then the plates were vacuum‐dried before storage at −20°C in a sealed plastic bag with desiccant until use.

Eu³⁺‐Labeled Antibody Preparation

Eu³⁺ labeling of monoclonal rabbit antihuman IgM was performed by using an Eu³⁺‐labeling kit strictly according to the manufacturer's instructions. Antihuman IgM (1 ml of 4.5 mg/ml) was loaded on a PD‐10 column and eluted using labeling buffer. Then the fractions were collected and concentrated to 2 g/l. Pipetted 500 μl of the obtained fraction purified the second antibody and mixed with 0.2 mg lyophilized Eu³⁺‐DTPAA, followed by vigorous stirring at 25°C for 20 h. The resulting mixture was then fractionated on a sepharose CL‐6B column (1 × 40 cm) and eluted with elution buffer. The absorbance values of the eluate were measured at 280 nm to obtain protein concentrations. After purification, equal amount of AR glycerin was added before it was subpackaged and stored at −20°C until use.

Specimen Preparation

Twenty‐five serum specimens from patients who fulfilled the clinical criteria for APS 2 and fifty normal ones from healthy volunteers referred to our hospital were collected. All serum specimens were separated at 2,360 g for 20 min at room temperature after collection and stored at −70°C for further analysis.

Detection of aCL IgM

Pipette 100 μl/well reference standards or serum diluted with buffer to microtiter plate coated with aCL antigen and the plate was incubated with shaking for 30 min at 25°C. After washing four times, the plate was then added to 100 μl/well Eu³⁺‐labeled antihuman IgM conjugate (1:20, the dilution buffer was 0.05 mol/l Tris‐HCl, pH 7.8, containing 0.9% NaCl, 0.2% of purified BSA, 0.01% Tween 20, 20 μmol/l DTPA, and 0.05% sodium azide as preservative) and incubated with shaking for 30 min at 25°C. It was washed again for six times and then pipetted 200 μl enhancement solution to each well and shook for 10 min before reading the fluorescent intensity by an auto‐DELFIA₁₂₃₅ TRFIA analyzer. Finally, the concentration of aCL IgM was calculated according to the calibration curve using the Multicalc software.

Evaluation on the Kits

1. Precision testing: Three pools of mixed serum specimens with high, intermediate, and low concentration of aCL IgM were subpackaged and stored at −20°C. The serum specimens were mixed and their concentrations were evaluated with samples, and the coefficients of variation (CVs) were calculated.

2. Linear range test: Serum from patient with the highest aCL IgM concentration was diluted in a twofold serial dilution manner. The serial dilutions were then detected by the established TRFIA kit and three commercial ELISA ones. The linear curve of dilution concentrations was obtained with dilution multiple as the horizontal abscissa and fluorescent intensity as the vertical ordinate.

3. Sensitivity test: The sensitivity of the assay was back‐calculated by the obtained mean fluorescent counts (n = 20) of the zero standard plus 2SD in the calibration curve.

4. Clinical applications: Sera from 50 healthy volunteers were collected to calculate the clinical specificity. Considering that the diagnosis of APS is complicated by the lack of a golden standard, results detected by the kits were correlated with the clinical criteria for APS.

5. Correlation test: The aCL IgM concentrations of 25 serum specimens from patients with APS was detected using the established TRFIA kit and an commercial ELISA one, respectively, and then the values obtained were analyzed.

6. Stability testing: The assay kits were placed into the 37°C water bath for 7 days and then the fluorescence intensities were compared with the one stored in routine conditions (4°C for 7 days).

7. Coefficient of recovery: Three sera with high antibody concentrations were diluted with sample buffer and assayed with the new TRFIA kit.

Statistical Analyses

Data were analyzed using the SPSS 13.0 software. Regression analysis and analysis of variance were used to calculate correlation and test linearity, respectively. Inter‐ and intraassay variation was calculated using the coefficient of variation.

RESULTS

Physicochemical and Immunological Identification of Eu³⁺‐Labeled Antibody

Eu³⁺‐labeled rabbit antihuman IgM was fractionated on a sepharose CL‐6B column and then the first elution peak was collected (Fig. 1). Using the Eu³⁺ standard provided by PE‐Wallac as reference, we calculated that the concentration of Eu³⁺ in the Eu³⁺‐labeled second antibody was 0.029 μmol/l and the protein was 0.0043 μmol/l; in other words, there were 6.74 Eu³⁺ combined to one molecule of rabbit antihuman IgM on average.

Figure 1.

Elution profile of Eu³⁺‐labeled rabbit antihuman IgM.

Calibration Curve for Determination of aCL IgM

As shown in Figure 2, the time‐resolved fluorescence intensities are directly proportional to the concentrations of aCL IgM. The standard curve of fluorescent intensity versus aCL IgM was found to be linear over this concentration range with a correlation coefficient of 0.999. The line equation for the calibration curve of aCL IgM was log(y) = 3.534 + 0.9404log(x), where x is fluorescence intensity and y is the concentration of aCL IgM (MPL U/ml). The sensitivity of the assay, defined as the mean signal of the zero standard plus 2SD, was 0.1 MPL U/ml.

Figure 2.

Standard curves for aCL‐IgM by TRFIA at different concentrations (4.85; 19.4; 77.6; 310.4; and 1,241.6 MPL U/ml). log(cps) = 3.534 + 0.9404log(conc), correlation coefficient = 0.999.

Linearity

A strong‐positive specimen was diluted from 2,630.9 to 0.08 MPL U/ml and the serial dilutions were detected with the established TRFIA as well as three commercial ELISA ones. The curve of detectable range of the two methods is shown in Figure 3, from which we observed that for the established TRFIA kit there was a good liner range within 0.16 to 2,630.9 MPL U/ml, whereas it was within 5.14 to 328.86 MPL U/ml when using the three commercial ELISA ones, indicating that the method of TRFIA we established has a wider detectable range than the commercial ELISA.

Figure 3.

Serial dilutions of a specimen with the highest aCL IgM concentration were tested with TRFIA and ELISA, respectively. Double y‐axis curve was determined according to aCL IgM concentration (x axis) as well as TRFIA fluorescence intensity and ELISA absorbance.

Assay Precision

The precision of the assay was also analyzed by measuring three pools of mixed serum specimens with high, intermediate, and low concentration of aCL IgM 20 times in one series (intraassay) and in duplicate in eight different series. As shown in Tables 1 and 2, the intraassay CVs detected by the established aCL IgM TRFIA at mean aCL IgM concentrations of 368.3, 72.9, and 13.8 MPL U/ml were 2.38, 3.27, and 3.93%, respectively, and the interassay CVs at mean concentrations of 368.7, 72.8, and 13.9 MPL U/ml were 2.91, 3.74, and 4.46%, respectively.

Table 1.

Intra‐Batch Precision Analysis (n = 20)

Group	Concentration (MPL U/ml)	SD	CV (%)
High	368.3	8.782	2.38
Intermediate	72.9	2.387	3.27
Low	13.8	0.542	3.93

Table 2.

Inter‐Batch Precision Analysis (n = 8)

Group	Concentration (MPL U/ml)	SD	CV (%)
High	368.7	10.733	2.91
Intermediate	72.8	2.725	3.74
Low	13.9	0.62	4.46

Correlation With ELISA

The concentrations of 25 serum samples from patients who fulfilled the clinical criteria for APS obtained by TRFIA were compared with those obtained with an ELISA kit. The scatter plots showed that the two methods correlated well (Fig. 4) and the correlation coefficient of the results obtained from the two assays was 0.9552.

Figure 4.

Correlation between the TRFIA and ELISA.

Clinical Application

The established TRFIA immunoassay was then applied to analyze the aCL IgM levels of serum specimens from 50 healthy volunteers and only one false‐positive case was produced, that is, the specificity of the assay was 98%.

Stability of the TRFIA Kits

The assay kit were put into the 37°C water box for 7 days, and we observed that it exhibited no visible change except for a decline by 15.37% in the rate of combination when compared with the one stored in routine conditions, suggesting that the established assay kit had good stability and could be stored at 4°C for 1 year.

Coefficient of Recovery

The recoveries of the first sample were 102.30, 97.57, and 109.06%, respectively. The recoveries of the second sample were 102.60, 104.78, and 105.76%, respectively. The recoveries of the third sample were 98.03, 96.43, and 93.69%, respectively. The average recovery rate was 101.13% (Table 3).

Table 3.

Coefficient of Recovery: Three Sera With High Antibody Concentrations Were Diluted With Sample Buffer and Assayed in the New TRFIA Kit

Sample	Dilution	Observed (U/ml)	Expected (U/ml)	O/E (%)
1	1:100	157.8
	1:200	78.23	78.9	99.15
	1:400	40.96	39.45	103.83
	1:800	19.57	19.725	99.21
2	1:100	135
	1:200	65.14	67.5	96.50
	1:400	35.68	33.75	105.72
	1:800	16.78	16.875	99.44
3	1:100	171.56
	1:200	89.76	85.78	104.64
	1:400	45.9	42.89	107.02
	1:800	21.88	21.445	102.03

DISCUSSION

Classic aCL test uses cardiolipin as antigen to coat on the polystyrene microtiter plates. However, direct antibodies to cardiolipin are not the pathogenic antibodies responsible for APS and the real “culprit” of the syndrome is indirect aPL directed against a host of phospholipid‐depending proteins, for example, β₂GPI 11, 12, 13. Though not as specific as the assay detecting a single or highly purified glycoprotein including anti‐β₂GPI, prothrombin, or phosphatidylserine/prothrombin complex in theory, aCL test is still the most useful approach to serological diagnosis of APS owing to its high diagnostic sensitivity and its combination with LA test should capture the majority of the underlying patients 14, 15, 16. Yet limitations inherent in the use of ELISA, the technique traditionally used for aCL detection, preserve the clinical utility of the test due to its poor analytical quality. As we all know, the laboratory proof of APS includes LA, aCL, or anti‐β₂GPI in medium or high titers (i.e., >40 GPL or MPL) on two or more occasions at least 12 weeks apart 2. Nevertheless, cutoff levers for negative, low, medium, and high titers remain contentious despite 20 years’ efforts at standardization and interpretation of aCL ELISA results. To better the detection of aCL IgM, the earlier serological hallmark for APS diagnosis, we develop in this study a new immunoassay based on TRFIA, which has been recently proposed as a more sensitive alternative to classical ELISA and an almost ideal way for quantification of analytes of clinical and biological importance 17 owing to its characteristics of wider detection range, higher sensitivity, and less susceptibility to matrix interference. According to the 13th International Congress on Antiphospholipid Antibodies 18, the complex of cardiolipin plus bovine β₂GPI was used as antigen and Eu³⁺‐labeled rabbit antihuman IgM as conjugate.

The precision test, which is a major concern for an assay kit, illustrated that the aCL IgM TRFIA we developed was reliable since its inter‐ and intraassay CVs were very small. Its reliability was further affirmed by a recovery test and the average recovery rate was 101.13%. The established TRFIA kit for aCL IgM detection has a good liner range within 0.16 to 2,630.9 MPL U/ml, whereas it was within 5.14 to 328.86 MPL U/ml when using three commercial ELISA ones, indicating that the method of TRFIA we established has a wider detectable range than the commercial ELISA. In our previous use of aCL IgM ELISA kit, we noticed its tendency to produce low‐positive results and critical ones that were difficult to confirm due to the poor precision or reproducibility of the assay; and this may obscure the true diagnosis of this part of the patients and unnecessary anticoagulant treatment may be given, and thus putting these falsely diagnosed patients at a high risk of bleeding and osteoporosis 19, 20, 21. The sensitivity of the aCL IgM TRFIA we established is 0.1 MPL U/ml, and this remarkable improvement in sensitivity makes it more suitable as a screen tool for APS since its lower detection limit is far below the normal upper limit of the reference value (6.93 MPL U/ml). On the other hand, the specificity of the new assay was 98%. Moreover, taking into account that the allowance of variations was greater by using fluorescence intensity than optical density (OD) as to impacting the same degree on the final concentration, the superiority in measurement accuracy of a TRFIA system to an ELISA one is obvious. In our study, we also noticed that the counts of fluorescent intensity obtained by the aCL IgM TRFIA kit varied little, even put the microplates overnight after the addition of enhance solution, whereas the ODs obtained by the commercial ELISA one decreased with time and could hardly put through 30 min after the addition of stop solution. This superiority in stability of TRFIA may be largely due to its independence of the disintegration of the labeling compound.

This is, to the best of our knowledge, the first report to develop a novel immunoassay based on a TRFIA system for aCL IgM detection and from the data presented above, we conclude that the most meaningful consequence of this work is that the aCL IgM TRFIA we developed gives promise to a more sensitive and reliable diagnosis of APS and has potential value for large‐scale screening programs.