Question

Compare and contrast the use of the following methodologies for diagnosing disease: ELISA, PCR, WESTERN BLOTS, and Fluorescent antibody techniques.

In your answer discuss the relative sensitivity and specificity of the assays.

Lastly, when might culturing methods provide useful information to the doctor or clinician that the other methods do not.

Answer 1

ELISA.

The LPS solution was diluted 1/100 in carbonate-bicarbonate buffer (pH 9.0). Ninety-six-well microtiter plates (Polysorb; NUNC, Wiesbaden, Germany) were coated with 50 μl of antigen for 1 h at 37°C. The wells were washed with PBS-Tween (0.05%; pH 7.2) and blocked with 75 μl of 10% goat serum in PBS-Tween. After another washing step, 50 μl of patient serum samples, as well as positive and negative controls, diluted 1/500 in 10% goat serum, was added and incubated for 1 h at 4°C. After the plates had been washed four times, 50 μl (each) of horseradish peroxidase (Sigma, Taufkirchen, Germany)-conjugated goat anti-human immunoglobulin G (IgG), IgM, and IgA (Gibco, Eggenstein, Germany), diluted 1/2,000 in 10% goat serum, was added and incubated for 1 h at 37°C. Goat anti-human immunoglobulins were found not to be reactive with Francisella antigen. After six rinses with PBS-Tween, a substrate reaction was started with 50 μl of 66% tetramethylbenzidine (TMB; Seramun, Dolgenbroth, Germany) and stopped after 10 min with 50 μl of 2.5 N sulfuric acid. OD405 values were determined by using an Asys Hitech microplate reader.

Enzyme-linked immunosorbent assays are among the methods of choice for large-scale investigations in the context of outbreak scenarios or epidemiological surveillance studies (4, 6, 28). LPS preparations immobilized on Polysorb microtiter plates were identical to those used for flow cytometry. Nevertheless, flow cytometry was obviously superior to ELISA in terms of test efficiency. The scatter graph in Fig. ​Fig.33 indicates that ELISA results for patient samples correlated significantly with mean fluorescence intensities obtained by flow cytometry. In contrast, ELISA results for the control sera spread over a wide range and did not correlate with the outcomes of corresponding measurements by flow cytometry. Dilution of sera down to 1/640 reduced the nonspecific background observed in control sera and increased the test efficiency of ELISA as assessed by ROC analysis. However, further dilution again led to an increased overlap of titers between patients and controls due to the decreased sensitivity of the assay (data not shown). A certain number of negative sera showed a relatively high background; however, these sera were probably overrepresented in this random sample. In larger studies (see “Seroepidemiology” below), the proportion of false-negative sera was much lower, even when a lower cutoff level was used.

Western blotting.

The LVS suspension was inactivated with 1% formalin overnight and adjusted to an OD560 of 2.5. Subsequently, 150 μl was treated with 50 μl of NuPAGE sample buffer (Invitrogen, Karlsruhe, Germany) and 10 μl of mercaptoethanol for 10 min at 70°C. After 15 min of boiling, the suspension was centrifuged for 20 min at 7,000 × g, and the soluble fraction was subjected to polyacrylamide gel electrophoresis (PAGE) at 130 V on a 4 to 12% NuPAGE gel (Invitrogen) for 1.5 h.

The gel was soaked for 10 min in transfer buffer (Novex, Frankfurt, Germany), and the bacterial antigens were transferred to a nitrocellulose membrane (Millipore, Billerica, Mass.) at 30 V for 1 h. The remaining binding sites on the membrane were blocked with 4% skim milk in Tris-buffered saline (pH 8.1) overnight at 4°C. The cut membrane strips were incubated with sera diluted 1/500 in 10% goat serum in PBS at room temperature for 2 h. For evaluation purposes, sera were diluted up to 1/2,000. After three rinses with washing buffer (Y.P. kit; Microgene, Munich, Germany), the strips were incubated with a polyvalent goat anti-human IgA-IgM-IgG horseradish peroxidase conjugate (Sigma) at room temperature for 1 h. Following another three rinses, the membrane strips were developed with precipitating TMB (Seramun). Sera were considered positive when they showed the typical LPS banding pattern at a dilution of 1/2,000.

TABLE 1.

Cutoff values, diagnostic sensitivity, and specificity of four approaches for the detection of tularemia-specific antibodiesa

Method	Unit	At 100% sensitivity	At 100% specificity
Cutoff	Specificity (%) (95% CI)	Cutoff	Sensitivity (%) (95% CI)
ELISA	Optical density	>0.648	98.0 (89.3-99.7)	>0.780	98.0 (89.3-99.7)
Flow cytometry	Mean fluorescence intensity	>1.59	100.0 (92.8-100)	>1.59	100.0 (92.8-100)
Indirect immunofluorescence assay	Intensity, titer	>1/80	92.0 (80.7-97.7)	>1/320	94.0 (83.4-98.7)
Microagglutination	Agglutination, titer	>1/16	100 (92.8-100)	>1/16	100.0 (92.8-100)
Western blotting	Visible LPS pattern, titer	1/2,000	100 (92.8-100)	1/2,000	100.0 (92.8-100)

As shown in Table ​Table1,1, Western blot analysis clearly distinguished between patients and controls. Dilution of sera down to 1/2,000 did not affect the appearance of the typical LPS banding pattern but eliminated nonspecific bands seen in some of the control sera. Cross-reactivity has been assumed for proteins homologous to the highly conserved chaperone proteins DnaK, GroEL, and GroES of Escherichia coli (17). Enzymatic digestion of the LPS extract decreased the amount of contaminating proteins. However, some heat shock proteins may have been resistant to proteinase K, since the respective bands remained visible in the Western blot after digestion.

Sample preparation for PCR assays.

Total nucleic acids were isolated from 200 μl of each respiratory specimen as previously described (13). To ensure that negative results were not due to poor nucleic acid extraction or inhibition of the PCR assay, 1,000 copies/PCR of the EXO specimen processing control, a 262-base RNA transcript derived from jellyfish DNA (16), were added to the lysis buffer. One low-positive control containing 200 to 1,000 copies/PCR of each respiratory virus harvested from cell culture and diluted in minimal essential medium and one negative control consisting of cultured, uninfected human epithelial cells were processed with each batch of clinical specimens.

TaqMan assays.

Specimens were tested for RSV, PIV types 1, 2, and 3, FluA, MPV, and AdV using seven separate, quantitative PCR assays. Samples were analyzed without knowledge of the patient's FA result. One hundred specimens (8.8%) were not tested for AdV due to insufficient sample volume. Although PCR tests were developed for FluB and PIV type 4, they were not used to test these specimens because only one sample each was positive by FA for FluB and for PIV type 4 during this period.

The RT-PCRs were performed for the RNA viruses using a one-step RT-PCR master mix as previously described (13). AdV DNA was detected using a PCR master mix (Quantitect multiplex PCR kit; QIAGEN, Inc., Valencia, CA) and the following cycling parameters: 50°C for 2 min, 95°C for 15 min, and 45 cycles of 94°C for 1 min and 60°C for 1 min. The PCR primer and probe sequences for detection of RSV (13) and MPV (14) have been previously described. The primers and 6-carboxyfluorescein (FAM)-labeled probes for detection of FluA, PIV1, PIV2, PIV3, and AdV were designed using Primer Express software (Applied Biosystems, Foster City, CA) from multiple aligned sequences of each virus obtained from the NCBI database. The primer and probe characteristics and sequences are given in Table ​Table1.1. The assays for RSV, FluA, PIV1, PIV3, and AdV were duplexed with a second primer set and VIC-labeled probe (13) that amplified and detected the exogenously added EXO specimen processing control.RNA transcripts for each RNA virus amplicon were synthesized, purified, and quantified as previously described (13). Contaminating DNA was not detected by PCR amplification in up to 106 RNA transcript copies. Tenfold serial dilutions of 107 to 10 copies of each RNA transcript for the RNA viruses and EXO were added to the RT-PCRs in duplicate. Similar dilutions were prepared using a plasmid containing the AdV amplicon sequences. The results were used to generate standard curves for quantification of the respiratory viruses and EXO in clinical samples. The threshold cycles of samples were compared to the standard curves; results were expressed as virus copies per milliliter of original sample. All samples with negative respiratory virus results required detection of at least 200 EXO copies per reaction to be considered valid. Nucleic acid extraction (if sufficient sample volume was available) and PCR were repeated on all samples that were negative for both respiratory viruses and EXO. Specimens that did not amplify EXO after repeat analysis were considered unsatisfactory for PCR.

Respiratory virus antigen detection (FA).

Specimens were tested for RSV, PIV (types 1 to 4), FluA, influenza type B (FluB), and AdV by use of an indirect fluorescent-antibody assay optimized to yield the most accurate and reliable results possible. After addition of an antibiotic solution and aspiration and expulsion with a Pasteur pipette to break up mucus, the respiratory specimens were centrifuged for 10 min at 4°C at 700 × g. If a pellet was visible, cells were dripped onto a 10-well slide and air dried. If no pellet was seen, a slide was prepared by cytocentrifugation. Additional washes were done if the cell pellet was too thick with mucus. The slides were fixed in acetone and incubated with virus-specific mouse monoclonal antibodies (Chemicon, Temecula, CA). Each lot of antiserum was titrated to its optimum dilution, and fresh reagents were prepared weekly. After a 30-min incubation at 37°C with primary antibody, goat anti-mouse fluorescein-conjugated monoclonal antibodies (ICN Biomedicals, Inc., Costa Mesa, CA) were applied to the sample wells, and the slides were incubated for an additional 30 min at 37°C. Slides were washed and read immediately using a fluorescence microscope. The presence of bright green fluorescence within intact cells was considered positive. Slides with too few intact cells were considered inadequate for analysis. Each FA slide was read twice by technologists with an average of 15 years of virology laboratory experience. This careful processing resulted in an interpretable FA slide in 95% of samples; approximately 1% of samples had a discrepant reading.

Results of respiratory virus detection by FA and PCR.

The results of FA and PCR on 1,138 specimens from children with respiratory illness are shown in Table ​Table2.2. One or more of the seven respiratory viruses was detected in 436 (38.3%) of the 1,138 respiratory specimens by FA and in 608 (53.4%) by PCR (P < 0.001), including 58 specimens in which only MPV was detected. Specimen quality was inadequate for FA due to insufficient cells in 52 (4.6%) of 1,138 specimens (1 swab, 3 tracheal aspirates, and 48 nasal washes). Of these 52 specimens, 13 (25%) were positive by PCR (6 for RSV, 2 for FluA, 1 for PIV3, 1 for AdV, 2 for MPV, and 1 for both PIV3 and MPV). Eighteen (1.6%) of the 1,138 specimens (all nasal washes) were considered unsatisfactory for PCR based on low EXO control amplification, 1 of which was positive for RSV by FA. Six (1.4%) of the 436 specimens positive by FA and 83 (13.7%) of the 608 specimens positive by PCR were positive for more than one virus. The nasal wash and swab specimens were more likely to be positive by either FA or PCR (426 [39.2%] and 590 [54.2%], respectively, of 1,088) than were the tracheal aspirates and BAL specimens (10 [20%] and 18 [36%], respectively, of 50) (P < 0.007).