CUBAN MEDICAL LITERATURE

Dengue IgM Detection UltramicroELISA Test
with Ready-to-Use Reagents

Regla de la Caridad Herrera(1); Carmen Acosta(2); Antonio Melchor(3); Vivian Alonso(3); Rosa Lidia Solís(4); Susana Vázquez(5)

ABSTRACT: Dengue is currently the most important human arbovirus. It is reported in more than 100 countries, and 2.5 billion people are at risk of infection. Annually, there are an estimated 50 million people infected, with more than 500,000 hospitalized, and 25,000 to 50,000 deaths. Because of the emergence and re-emergence of dengue and hemorrhagic fever, the diagnosis of this disease by laboratory tests to distinguish dengue from other diseases, as well as to support surveillance systems, is of upmost importance. The best choice for the diagnosis of dengue fever is an immunoglobulin M-specific capture enzyme-linked immunosorbent assay (ELISA), due to its specificity, sensitivity, and fast performance. An example of this technique is IgM dengue ultramicroELISA. We present the standardization results of dengue ultramicroELISA’s new format, which includes strip plates and ready-to-use reagents. The original conditions of coating and conjugate dilution of the UMELISA Dengue IgM kit were maintained. Both within-run and between-run precision values were below 10%; the stability of all reagents was higher than 90%. There were no significant differences when comparing the previous reagents and plates with the new ones. Compared to hemagglutination inhibition, the sensitivity was 94.3%, the specificity was 94.1%, and the kappa concordance index was 0.91.

Results indicated the ultramicroELISA’s new format maintains its reliability for early detection of dengue.

Keywords: DENGUE; ULTRAMICROELISA; IGM; DIAGNOSIS

INTRODUCTION

Dengue is an infectious disease caused by an RNA virus of the Flaviviridae family. There are four known serotypes (DEN-1, DEN-2, DEN-3 and DEN-4).[1] It is transmitted by the Aedes aegypti mosquito as its main vector.[2] In terms of morbidity, mortality and economic cost, it is the most important arbovirus globally, with an estimated 100 million cases annually.[3,4]

In acute dengue infections, two patterns of serum response can be observed: primary and secondary. The first is observed in persons who are not immune to flavivirus. In this response, IgM antibodies increase between the third and fifth day after onset of the symptoms, reach a peak at two weeks and at three months, are reduced to practically undetectable levels, although they may persist up to 90 days in some cases. The IgG antibody increase takes place between the 10th and 14th days, where they remain for life. The secondary response is observed in persons with an acute dengue infection that have previously suffered a flavivirus infection. In this case, IgG antibody titers rise to high levels between the first and second day, while the IgM antibodies rise more slowly and to a lower level than in primary infections.[5-9]

Most primary infections result in dengue fever (DF), a mild disease characterized by biphasic fever, intense headache, myalgia, arthralgia, skin rash, lymphadenopathy and leucopenia.[10] The severe forms of the disease, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), are usually associated with secondary infections.[11] DHF is characterized by high fever, associated hemorrhagic phenomenon with reduction of the number of platelets, increase in capillary permeability and hepatomegaly. In DSS, the state of the patient suddenly deteriorates and signs of circulatory failure appear.[12]

Despite advances in the knowledge and clinical epidemiological characterization of DF, DHF and DSS, laboratory support is essential in the differential diagnosis by virus isolation, serological diagnosis or molecular detection. Routine serological diagnosis includes hemagglutination inhibition (HI), complement fixation (CF), neutralization (N) and IgM capture ELISA (MAC-ELISA).[13]

The dengue IgM ultramicroELISA (UMELISA) is an example of MAC-ELISA.[14] With the objective of improving its use and manipulation, the kit format was modified to strip plates and ready-to-use reagents. Results in the standardization of the new format are presented in this study.

MATERIALS AND METHODS

Samples of human sera: Fifty-two pairs of sera from patients clinically suspected of dengue infection were evaluated. The samples were collected during the dengue epidemic that took place in the first semester of 1997 in Santiago de Cuba, Cuba.[15,16] The sera were preserved at -20ºC. They were classified into primary infection (n = 4), secondary infection (n = 31), and not dengue (n = 17), according to HI criteria.

Solid Phase: Rigid ultramicroELISA (10 ìl per well, 96 wells) plates and strips (8 wells x 12 strips; Greinerlabortechnik, Germany) were used.[17]

Coating antibodies: Human anti-IgM antibodies taken from sheep (Center for Immunoassay), were adsorbed on AH-Sepharose 4B-human IgG and purified by affinity chromatography on AH-Sepharose 4B-human IgM. They were finally dialyzed with a phosphate buffered saline solution (PBS) pH 7.4, and stored in the same solution at -20°C until use.

Control sera: The negative control serum was obtained at the Marianao Provincial Blood Bank, City of Havana, Cuba, from a blood donor. To prepare the positive control serum, dengue-infected patient samples from Brazil and Colombia were mixed. The positive or negative of the control sera was evaluated by the HI technique and MAC-ELISA of the Pedro Kourí Institute of Tropical Medicine (IPK), Cuba.

Antigen: For the HI and UMELISA tests, antigens, extracted by the acetone-saccharose method[18] from brains of suckling mice inoculated with dengue 1 (Hawaii), dengue 2 (New Guinea), dengue 3 (H-87) and dengue 4 (H241) were used.

For the HI test, only serotypes DEN1 and DEN2 were used, and for the UMELISA, a mixture containing all four serotypes, each one having 16 hemagglutinating units (HU), was used.[19]

Enzyme-antibody conjugate: A dengue-complex monoclonal antibody (IgG1 isotope), purified by affinity chromatography using a Protein A-Sepharose CL 4B column was conjugated with alkaline phosphatase by the one-step glutaraldehyde method.[20]

Fluorigenic sustrate: A concentrated solution (0.5 mM) of 4 metilumbelliferil phosphate (Koch Light Ltd. Haverhill, Suffolk, Great Britain) was used. Diethanolamine (1M, pH 9.8) ( Merck, Germany) was used for its dilution.

Equipment: A MAS 301 washer was used for the plates and a PR-521 Photometer-Fluorimeter was used to measure fluorescence intensity. This equipment uses SUMA technology.[21]

HI test: The original method, adapted to a micro technique format with 8 HU of each virus, was used.[18] Paired sera were considered positive when there was a four times titer increase between the first and second serum. Cases in which one of the sera presented 1:2560 titers were considered secondary infection.[1]

DENGUE IgM UMELISA: This is a heterogeneous immunoenzymatic test of the capture type. Three one-hour incubations, each at 37ºC, were done to the samples and control sera, the antigen and the conjugate. After each incubation, 6 washing cycles were performed with 15 mM Tris-HCl, 0.05 % Tween-20 buffer, pH 7.8. Finally, the substrate was added and incubated for 30 minutes at room temperature. The results were expressed in fluorescence units (FU) and calculated as the relation M-B/P-B, where M, B (sample diluting solution) and P were respectively the sample, blank and positive control serum fluorescence units. The samples for which the relation value was higher or equal to 0.3 were considered positive.

Solid Phase coating: Rigid plates (control plates) and the strips (test plates), were coated with different concentrations of human anti-IgM in 10 mM Na2CO3/NaHCO3, pH 9.6. They were incubated during 4 hours at 45°C and were finally blocked with PBS containing 0.1% bovine seroalbumin (BSA).

Preparation of the control sera: Two variants were used: concentrated (control variant) and ready-to-use (test variant). At the time of the test, the concentrated control was diluted 1:21 with a sheep serum solution in 5% 15mM Tris-HCl, 0.05% Tween-20, pH 7.8; while ready-to-use sera were provided in the 1:21 dilution and used directly as supplied in the kit.

Preparation of the conjugate: As for the control sera, two variants were used: a concentrated (control variant) and a ready-to-use one (test variant). In the first case, the conjugate working dilution was prepared just before carrying out the test. Meanwhile, in the ready-to-use variant, a ready-to-use conjugate solution was supplied in the test kit.

Selection of the test conditions: The use of strip plates required a reevaluation of the coating concentration and the conjugate dilution. A crossed titration was done for the simultaneous evaluation of different coating concentrations (from 4.5 to 17.5 ìg/ml of human anti-IgM) using different conjugate solutions (from 1:1000 to 1:9000).

To select the optimal combination of coating concentration and conjugate dilution, the selected concentration had to be within the plateau zone, which is defined as the range of concentrations for which the FU of each control sera remained constant. And there had to be an adequate difference between the control sera, values under 10 FU for the negative, and between 130 and 150 FU for the positive.

Reproducibility Study: In this study, the 2 solid phases were used and 5 samples with different positive levels. The samples were applied 10 times in each plate in five consecutive days to determine the within-plate and between-plate variation coefficients (VC).

Shelf stability: This study was carried out on the strip plates and the ready-to-use control sera and conjugate. The results obtained on the initial day of the study (time zero) for the control sera, under each of the analyzed conditions, was considered the control condition, while the test condition was the monthly results during the following 24 months. The stability criterion was maintained as long as no significant differences were observed. Storage temperature was 4ºC.

Comparison of the plates, the control sera and the conjugate: A comparison was made between the strip and rigid plates and the ready-to-use control sera and conjugate with respect to the concentrated ones, using 20 replicas of each.

Comparison of HI and the new UMELISA format: Using the results of the 52 paired sera, the specificity and sensitivity of the new Dengue IgM UMELISA format was determined with respect to the HI test, as well as the concordance between both tests by the kappa statistic.[22] The capacity of one or the other technique to diagnose dengue infection using the first serum in the pair (S1), taken during the acute disease phase, was also evaluated.

Statistical Analysis: In the shelf stability test, the comparison was performed by the X 2 test. The comparison of plates, control sera and conjugate was done using the Student t test. Both tests were carried out for a= 0.01 using the Microsoft Excel 2002 program.

RESULTS

Selection of test conditions

Using the strip plates, both the coating concentration (12.5 ìg/ml human anti-IgM) and the conjugate dilution (1:7000) agreed with the ones used for the rigid plates. In Figures 1 and 2, control sera results are shown for the different coating concentrations evaluated and the 1:7000 conjugate in both types of plates. Figure 3 presents the control sera results for both plates using 12.5 ìg/ml human anti-IgM and different dilutions of the conjugate tested.

Figure 1:Evaluation of different coating concentrations in the rigid plates using the positive control serum (PCS) and the negative control serum (NCS)

Figure 2: Evaluation of different coating concentrations in the strip plates using the positive control serum (PCS) and the negative control serum (NCS)

FIGURE 3: Evaluation of different conjugate dilutions in the rigid and strip plates using positive (PCS) and negative (NCS) control sera

Reproducibility Study

The within-plate and between-plate variation coefficients did not exceed 4% and 7%, respectively (Table 1).

TABLE 1: Reproducibility results using rigid and strip plates

Shelf Stability

Throughout the study, the FU results of the control sera did not show significant differences with respect to time zero (data not shown). Table 2 shows the averages of the FU obtained at time zero and after 24 months.

TABLE 2: Results of the shelf stability test for the strip plates using control sera and conjugate

T0: Time zero evaluation
T24: Evaluation after 24 months
%: Percentage of recovery
24 months after time zero

Comparison of HI and the New UMELISA Format

The distribution of the results using the new UMELISA format and the HI are shown in Table 4. Using the paired sera classified as not dengue by the HI test, there was concordance in 16 out of 17, while one pair (number 21) was evaluated as positive by UMELISA. Using this test, 4/4 pairs of primary infection cases were detected and 29/31 secondary infection cases. In this test, the discordant pairs were numbers 12 and 50, diagnosed as negative with UMELISA. The data for the differing samples are shown in Table 5. The S1 samples used to study the capacity of both tests for diagnosing dengue infection using only an acute phase serum were obtained between day 1 and 5 of symptom onset, when the patients first went to the doctor. The UMELISA test detected 2 cases of primary infection with S1 sera, while none was detected by the HI. In the sera pairs of secondary infection cases, the UMELISA test was capable of establishing the diagnosis of infection in 11, which were included in the 13 detected by HI.

TABLE 3: Averages and Student t test comparison

RU: Ready-to-use reagent
t (: 0,01) = 2.86

TABLE 4: Distribution of positive and negative sera by HI and UMELISA

TABLE 5: Results of the discordant samples

The sensitivity, specificity and kappa index were 94.3%, 94.1% and 0.91, respectively.

DISCUSSION

The Pan American Health Organization (PAHO) states that every country endemic for dengue must have a passive surveillance system based on clinical diagnosis, which considers dengue a notifiable disease.[6] However, since it is frequently impossible to clinically differentiate dengue from other diseases[23] caused by several viruses, bacteria and even some protozoa, by the time cases are detected and reported by doctors, a considerable transmission of dengue has already taken place that may have even reached the maximum.

The similarity of clinical symptoms to those caused by other pathogens, as well as the increase in the prevalence and incidence of dengue fever, dengue hemorrhagic fever and dengue shock syndrome,[24] have made clear the importance of an active surveillance based on laboratory tests. With this surveillance, the necessary information regarding onset, location, viral serotype and the severity of the disease may be obtained.[25]

The techniques generally used for dengue diagnosis are HI[18] and ELISA to detect IgM or IgG antibodies.[26] The HI test, as well as the IgG ELISA, requires paired samples collected with an interval of at least 7 days for a final diagnosis when a four times or greater titer increase is found.[6] On the other hand, the IgM ELISA (MAC- ELISA), can frequently diagnose the disease with only one acute phase serum. Several studies have demonstrated that IgM antibodies are detectable between the sixth and tenth day in 95% of the cases, whether primary or secondary.[27] For this reason, IgM capture ELISA is presently the most useful technique for active surveillance and its use has become widespread in recent years.

At present, the development and production of different reagent kits based on MAC-ELISA is being promoted,[24] the Dengue IgM UMELISA among them. Its original format consisted of rigid plates (96 wells), lyophilized antigen and concentrated control sera and conjugate. Considering that the current trend in kit formats is directed towards using lyophilized antigen and the remaining reagents in ready-to-use form,[28] the objective of this work was to evaluate the substitution of the concentrated control sera and conjugate by ready-to-use ones, as well as analyzing possible replacement of the rigid plates by strips.

During the evaluation of the coating concentration and the conjugate dilution in the strip plates, the coating plateau started at 10 ìg/ml, that is the fluorescence values for each of the control sera remained unchanged. Considering that other researchers have stated,[29] that it is necessary to work in the coating plateau zone, the value selected as optimal concentration was 12.5 ìg/ml. This concentration is in the zone mentioned, and besides, by using this concentration and the 1:7000 conjugate, it was possible to obtain a good separation between the positive control serum and the negative one (8 and 137 FU, respectively). Thus, it was not necessary to change the previous coating and conjugate concentrations.

With respect to reproducibility, the variation coefficients within-plates and between-plates did not exceed 10%, indicating that there was adequate precision, which guarantees the homogeneity of the results for the same sample evaluated at different times.[30]

The statistical analysis of the stability results evidenced that the strip plates as well as the ready-to-use control sera and conjugate were stable during 24 months when preserved at 4ºC. This result showed that their introduction did not alter the stability of the reagents with respect to the original Dengue IgM UMELISA kit format (18 months at 4ºC).

In the evaluation of the plates – in which no significant differences were found when comparing the strip to the rigid ones - showed that the results were equally reliable if the previous conditions of time and storage temperature were maintained. This result is of vital importance since it increases the possibilities of UMELISA use. The introduction of strip plates allows the kit to be used both in massive screening laboratories, as well as in those where a small number of samples is processed, without wasting determinations or delaying the diagnosis waiting for the number of samples (90) required to complete the rigid plates.

When the ready-to-use control sera and conjugate were compared with the concentrated reagents, there were no significant differences either, so it is possible to eliminate the final dilution step in the preparation of these reagents, thus eliminating possible changes in the test derived from their preparation.

In the analysis of the samples by UMELISA and HI, we obtained three pairs of sera for which results differed. The first discordant pair was number 21, classified as not dengue by HI (titers below 1:1280, without seroconversion),[31] and weakly positive by UMELISA. This result could be due to the higher sensitivity of the ELISA techniques with respect to HI.[32] The other two discordant pairs were numbers 12 and 50, which showed seroconversion by HI and the titer of the convalescence serum was 1:2560, compatible with a secondary infection.[31] In such cases, a small number of persons have been reported in which IgM antibodies may not be detected.[33,34]

Considering that one of the main disadvantages of the HI is the need for paired sera to carry out dengue infection diagnosis (except in those cases in which the first serum has titers over 1:2560), the capacity of both techniques to carry out the diagnosis with sample S1 was evaluated as part of the comparison between UMELISA and HI. Neither of the techniques, UMELISA or HI, was particularly efficient in dengue infection diagnosis using the acute phase serum. Of the 33 paired sera evaluated as positive by HI, UMELISA diagnosed 31% of them using the acute phase serum (13/33).

Two of them were from secondary infection and 11 from primary infection cases. On the other hand, HI allowed reaching a diagnosis in 13 samples of secondary infection. The two primary infection samples that were not detected by UMELISA were taken on the first and second day of symptom onset, earlier than the time required for IgM antibodies to reach detectable levels in primary dengue infections.[35] In the secondary infection cases, HI excelled UMELISA, which could be because HI detects total antibodies[18], IgM, as well as IgG. The latter predominate in this type of infection and that is why in reinfections they are considered the gold standard of markers.[28]

The agreement between the new UMELISA format and the HI was 94.2%, similar to the 92.85% when the original format is compared to HI.[14] This result reaffirms that the format change does not have any effect on the previously established kit quality.

The kappa index (0.91) showed good concordance with the HI.[22] The percentages of sensitivity (94.3%) and specificity (94.1%), were comparable to those of other diagnostic kits designed for the same purposes[33] and allowed the application of the test for massive screenings in populations suspected of being infected by dengue.

CONCLUSIONS

The UMELISA Dengue IgM kit with strip plates and ready-to use reagent format improves present conditions of manipulation and use. This test, performed in four hours, is a favorable alternative for the serological diagnosis of dengue infection from a single serum sample and does not have any of the HI limitations.

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This paper was first published in Revista Biomédica 2005;16:1-13, Jan-March, 2005, Universidad Autónoma de Yucatán, México.

THE AUTHORS

1. Infectious Diseases Laboratory, Center for Immunoassay, Cuba
2. Monoclonal Antibody Laboratory, Center for Immunoassay, Cuba
3. Protein Purification Laboratory, Center for Immunoassay, Cuba
4. Finlay Institute, Cuba
5. Virology Laboratory, Pedro Kourí Institute of Tropical Medicine, Cuba