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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY Mar 2004 p 287 291 Vol 11 No 2 1071 412X 04 08 00H110010 DOI 10 1128 CDLI 11 2 287 291 2004 Copyright 2004 American Society for Microbiology All Rights Reserved Recombinant Protein Based Enzyme Linked Immunosorbent Assay and Immunochromatographic Tests for Detection of Immunoglobulin G Antibodies to Severe Acute Respiratory Syndrome SARS Coronavirus in SARS Patients Ming Guan 1 Hsiao Ying Chen 1 Shen Yun Foo 1 Yee Joo Tan 2 Phuay Yee Goh 2 and Shock Hwa Wee 1 Genelabs Diagnostics Pte Ltd 1 and Collaborative Anti Viral Research Group Institute of Molecular and Cell Biology 2 Singapore Republic of Singapore Received 10 November 2003 Returned for modification 11 December 2003 Accepted 29 December 2003 An enzyme linked immunosorbent assay ELISA and a rapid immunochromatographic test for detection of immunoglobulin G IgG antibodies in severe acute respiratory syndrome SARS patients were developed by utilizing the well characterized recombinant proteins Gst N and Gst U274 The ELISA detected IgG antibodies to SARS CoV in all 74 convalescent phase samples from SARS patients while weakly cross reacting to only 1 of the 210 control sera from healthy donors This finding thus led to a kit sensitivity specificity and accuracy of 100 99 5 and 99 6 respectively The test thus provided a positive predictive value PPV of 98 7 and a negative predictive value NPV of 100 In addition the ELISA gave a positive delta of 5 4 and a negative delta of 3 6 indicating an excellent differentiation between positives and negatives The same recombinant proteins were also applied to a newly developed platform for the development of a 15 min rapid test The resulting rapid test has an excellent agreement of 99 6 with a kappa value of 1 00 with the ELISA Again this rapid test was able to detect 100 of the samples tested n H11549 42 while maintaining a specificity of 99 0 n H11549 210 The PPV and NPV for the rapid test thus reached 95 3 and 100 respectively Severe acute respiratory syndrome SARS is a newly emerged disease of global significance because of its highly contagious nature in an increasingly globalized world This was promptly recognized by the World Health Organization WHO when the SARS epidemic spread beyond its place of origin in Guangdong Province China and a global alert was declared for the first time in WHO history The outbreaks affected over 8 098 people and spread to more than 29 coun tries and regions in a short period of 6 months with a mortality rate of up to 15 WHO summary report available at http www who int csr sars country table2003 09 23 en Obvious ly this disease is not a limited problem but one with profound consequences affecting sectors beyond public health care Although it is now understood that a novel coronavirus SARS CoV is the etiological agent for SARS 3 5 7 10 more remains to be learned in terms of how to detect diag nose and treat the disease Prompt identification and isolation of infected patients remain of paramount importance for dis ease control since no drug or vaccine is available for this disease With few options in the laboratory for detection initial disease management relied on travel and contact history and presentation of symptoms PCR tests developed with unprec edented speed are now available and can detect specific RNA of SARS CoV thus providing a laboratory method for diag nosis of SARS WHO had based on this method revised its criteria for case definition However existing protocols for PCR were not without their limitations The Singapore expe riences showed that the positive predictive values PPVs pro vided by PCR varied from 56 7 to 83 8 when different clinical specimens were used A E Ling SARS the Singapore lab oratory experience presented at the WHO Conference on SARS Research Singapore Republic of Singapore 19 June 2003 In a very recent report 13 the reverse transcription PCR RT PCR protocols of two WHO SARS network labo ratories were evaluated and the findings confirmed similar shortcomings of RT PCR Therefore the existing PCR cannot rule out the presence of the SARS virus when a negative result is obtained neither can it exclude the possibility of false de tection due to laboratory contamination 9 In the meantime methods involving indirect immunofluorescence assay IFA classic tissue culture for viral isolation and electronic micros copy of the cell culture for diagnosis are either time consuming or technically very demanding Alternative approaches are therefore critical for efficient management of the disease The enzyme linked immunosorbent assay ELISA is one such alternative because it can provide diagnostic information that is complementary to the data provided by PCR In addi tion a rapid point of care test such as an immunochromato graphic test can also facilitate case diagnosis and disease man agement in remote areas where laboratory facilities are not readily available In this study we were able to demonstrate the potential of two serological tests developed by utilizing two recombinant proteins Gst N and Gst U274 As reported else where Gst N was derived from the nucleocapsid region where as Gst U274 was derived from a unique protein of SARS CoV 12a Our study results suggested that the two serological tests each with an excellent sensitivity of 100 could provide Corresponding author Mailing address Genelabs Diagnostics Pte Ltd 85 Science Park Dr 04 01 Singapore Science Park Singapore 118259 Republic of Singapore Phone 65 67750008 Fax 65 67754536 E mail guanming sg 287 on March 17 2015 by MAHIDOL UNIV FAC OF MED http cvi asm org Downloaded from confirmatory information for the diagnosis of SARS especially for suspected cases initially screened by PCR as negative MATERIALS AND METHODS Recombinant proteins The materials and methods used for obtaining the recombinant proteins are described in detail elsewhere 12a Briefly both Gst N and Gst U274 used in the study were expressed as Gst fusion proteins in Esch erichia coli and purified with glutathione Sepharose beads Amersham Pharma cia Protein Gst N was derived from nucleocapsid of the SARS CoV with a highly conserved motif amino acids 111 to 118 found in all coronaviruses 12 deleted whereas Gst U274 was from a unique SARS CoV protein For this study the Gst U274 protein was further purified with a Superdex S 200 HR10 30 column on an AKTA fast protein liquid chromatography FPLC system Am ersham The buffer used contained 20 mM Tris HCl pH 7 5 100 mM NaCl 6 M urea and 1 mM H9252 mercaptoethanol the flow rate was 0 5 ml min and fractions of 1 ml were collected Fractions 12 and 13 were combined and dialyzed against phosphate buffered saline PBS overnight with at least three changes of buffer Serum specimens Seventy four convalescent phase serum samples were col lected from SARS patients admitted to the Tan Tock Seng Hospital or the Singapore General Hospital Ninety one control serum samples were obtained from healthly local donors who worked at the Institute of Molecular and Cell Biology Singapore Republic of Singapore All specimens were collected with consent and patient samples were collected at 16 to 65 days from the onset of symptoms In addition 119 sera from healthy donors purchased from BioClinical Partners Inc Franklin Mass were included in the study as additional healthy controls ELISA The Gst N and GstT U274 proteins were prediluted in carbonate buffer pH 9 6 at final concentrations of 0 1 and 0 15 H9262g ml respectively prior to plate coating The plates were prepared as described in reference 4 Briefly the 96 well polystyrene microtiter plates Immuno 1B Themo Labsystem Frank lin Mass were coated with the protein mixtures at a volume of 100 H9262l per well by incubation overnight 16 to 18 h at room temperature The plates were washed five times with PBS Tween 80 PBST and nonspecific binding sites were blocked with 200 H9262l per well of a Tris based diluent for1hatroom temper ature The plates were further washed another five times before 10 H9262l of serum in 200 H9262l of Tris based diluent containing 1 each bovine serum albumin BSA and skim milk powder was added Subsequently the plates were incubated for 30 min at 37 C followed by six washes with PBST Horseradish peroxidase conjugated goat anti human immunoglobulin G IgG 1 500 dilution was added at 100 H9262l per well and this mixture was incubated for 30 min at 37 C The plates were then washed six times in PBST and color development proceeded with the addition of the enzyme substrate tetramethylbenzidine TMB at 100 H9262l per well After a 15 min incubation in the dark at 37 C the reaction was stopped by adding 100 H9262l of 1 N HCl per well The optical densities OD were measured at 450 nm with a 620 nm reference filter Rapid immunochromatographic test The membrane based immunochro matographic test device consisted of a chromatography strip a separator and an absorbent pad all housed in a cassette as described previously 6 The chromatography strip was prepared separately according to the procedure detailed in reference 6 with slight modification before assembly into the device Briefly a nitrocellulose membrane with an average pore size of 8 H9262m Whatman Ann Arbor Mich was sprayed with the Gst N and the Gst U274 recombinant antigens in two separate lines at concentrations of 0 1 and 0 15 mg ml respec tively with a BioDot Irvine Calif spraying machine The membrane was dried for 10 min before being immersed for 1 min in a blocking buffer consisting of Milli Q purified water with 6 7 StabilCoat SurModics Inc 0 05 Triton X 100 and 0 5 casein The blocked membrane was then dried at 37 C for 60 min before being affixed to a membrane backing The reagent bearing pad was prepared using a porous matrix The porous matrix was sprayed with goat anti human IgG antibodies that were labeled with colloidal gold particles 25 to 30 nm in diameter This reagent bearing pad was then dried at 37 C for 2 h prior to incorporation into the device A chromatographic card was prepared by affixing a 0 1 Triton X 100 treated porous matrix to one end of the nitrocellulose strip and the reagent bearing pad to the other end on the same membrane backing This assembly was then cut into a strip approximately 4 by 56 mm 2 in size An assay device was assembled by placing an absorbent pad at the bottom half of the cassette and then a separator followed by one unit of the chromatographic strip before closing the top half of the cassette In addition a reagent releasing wash buffer was also prepared with Milli Q purified water with 50 mM NaH 2 PO 4 300 mM NaCl and 0 1 sodium dodecyl sulfate pH 8 0 When testing 25 H9262l of undiluted serum sample was added to the specimen window of the assay device The sample was allowed to migrate laterally and cover part of the membrane When the sample reached the indicator in the viewing window in approximately 30 s three drops of reagent releasing washing buffer were added to the buffer window to release the colloidal gold labeled goat anti human IgG antibodies The separator was then removed by pulling the protruding end to allow the chromatographic element and the absorbent pad to come into contact The colloidal gold labeled goat anti human IgG antibodies were then allowed to migrate across the chromatographic strip completely The result can be read in typically 2 to 15 min through the viewing window A negative result will be indicated by the appearance of a control line only whereas with a positive result the control line and either or both of the test lines appear in the viewing window Fig 1 Statistical analysis The kappa statistic was used to measure the strength of agreement between the results by the new rapid test and the new ELISA Kappa statistic values of H110220 75 0 40 to 0 75 and H110210 40 represent excellent agreement good to fair agreement and poor agreement respectively 11 For the data analysis for ELISA an arbitrary cutoff value of 0 45 was selected but confirmed with the delta values 2 which could provide an indication of an optimal differentiation between the positive and negative populations RESULTS ELISA When tested at a sample dilution of 1 20 the ELISA detected IgG antibodies to SARS CoV in 100 n H11005 74 of the convalescent phase samples from the SARS patients with a mean H11006 standard deviation OD value of 2 32 H11006 0 54 Table 1 and Fig 2 In contrast only 1 initial positive sample was found by the test out of the 210 control sera from the healthy donors with a mean H11006 standard deviation OD value of 0 05 H11006 0 05 FIG 1 Examples of the assembled rapid immunochromatographic test devices with their separators transparent tabs at the removed after assay position To the left is a device after an assay with a sample from a patient and to the right is another after an assay with a sample from a healthy control The three lines in the viewing window for the positive sample represent from top to bottom the control Gst N and Gst U274 respectively The negative sample generated the control line only 288 GUAN ET AL CLIN DIAGN LAB IMMUNOL on March 17 2015 by MAHIDOL UNIV FAC OF MED http cvi asm org Downloaded from P H11021 0 001 Fig 2 The test thus provided a PPV of 98 7 and a negative predictive value NPV of 100 Table 1 In addition the ELISA gave a positive delta of 5 4 and a negative delta of 3 6 indicating excellent differentiation between posi tives and negatives Furthermore and as shown in Fig 3 when titration curves were obtained with seven samples from the SARS patients by using this new ELISA the reactivity end point was found at dilutions ranging from H110221 40 to 1 640 with different samples Rapid immunochromatographic test When tested with un diluted samples the Gst N and the Gst U274 proteins used in the rapid test reacted to IgG antibodies in 100 42 of 42 and 85 7 36 of 42 respectively of the sera from SARS patients who met the WHO criteria for SARS Table 1 Thus the overall detection rate for the new test was 100 42 of 42 Table 1 Only the Gst N protein in the rapid test was found to cross react with 2 of the 210 sera from the healthy controls The test was therefore shown to have specificity PPV and NPV of 99 0 95 3 and 100 respectively with the tested populations Table 1 When the results were compared the rapid test and the ELISA gave an excellent agreement of 99 6 with a kappa statistic of 1 00 Table 2 In addition an excellent correlation R 2 H11005 0 988 was found when the reac tivity end points of the rapid test at dilutions ranging from 1 8 to 1 128 and the ELISA were compared by using the same seven patient samples Fig 4 DISCUSSION Recent studies have suggested the potential of a SARS CoV nucleocapsid protein as a diagnostic reagent for the detection of SARS 8 12a However the application of this protein in developing a viable diagnostic test remained to be fully dem onstrated The follow up study presented here provides results to fill in the gap The newly developed ELISA in this study utilizing the two Gst N and Gst U274 recombinant proteins was able to detect all 74 samples tested This result thus has demonstrated the capability of the new test for the detection of IgG antibodies to SARS CoV using the convalescent phase sam ples H1135016 days from the onset of the disease In addition the new test was found to be highly specific with only 1 initial false positive out of the 210 control subject samples tested Furthermore the reactivity end points obtained with the ELISA suggested satisfac tory sensitivity by a different parameter Although the results were not obtained with identical samples from a previous preliminary study Y C Wang SARS Diagnostics Scientific and Regulatory Challenges Workshop 2003 U S Food and Drug Administra tion http www fda gov ohrms dockets dockets 03n0281 03n0281 htm the range of dilutions from H110221 40 to 1 640 for the reactivity end points is nevertheless a good indication of sensitivity compa rable to if not better than those previously studied All of these findings suggested that the ELISA be ready for application at TABLE 1 Performance of individual markers and the new serological tests with sera from SARS patients and healthy donors Test format SARS patients Healthy controls PPV NPV No positive total Sensi tivity No positive total Speci ficity ELISA combined a 74 74 100 1 210 99 5 98 7 100 Rapid test Gst N 42 42 100 2 210 99 0 95 3 100 Gst U274 36 42 85 7 0 210 100 100 97 2 Combined 42 42 100 2 210 99 0 95 3 100 a Gst N plus Gst U274 FIG 2 Scatter chart of OD values obtained with sera from both SARS patients and healthy controls tested for IgG antibody to SARS CoV by using the newly developed ELISA FIG 3 Titration curves of seven SARS patient samples obtained with the ELISA TABLE 2 Agreement between the new rapid test and the new ELISA with sera from SARS patients and healthy controls Rapid test No of ELISA results Agreement Kappa statistic a Positive Negative Positive 43 1 99 6 1 00 Negative 0 208 100 a A kappa statistic of H113500 75 represents excellent agreement 0 40 to 0 75 represents good to fair agreement and H110210 40 represents poor agreement 11 VOL 11 2004 SEROLOGICAL TESTS FOR SARS 289 on March 17 2015 by MAHIDOL UNIV FAC OF MED http cvi asm org Downloaded from least for the exclusion of SARS in testing patients as advocated by the Centers for Disease Control and Prevention CDC The CDC recommended that the case definition for exclusion of SARS include the absence of antibody to the virus in convales cent phase serum obtained H1102221 days after onset of illness 1 The same recombinant proteins of Gst N and Gst U274 used in ELISA were also applied to the rapid immunochro matographic test but were applied separately as two different testing markers Intrinsically the newly developed rapid im munochromatographic test could provide not only indications for case detection but also details of maybe clinical signifi cance should a correlation be established later Although the biological function of the unique protein is yet to be unraveled the Gst U274 recombinant was found to be serologically reac tive and unique to SARS CoV 12a In addition the protein was found in some cases to give a higher reaction signal when tested for the IgM antibodies to SARS CoV 12a Because of this kind of finding the usefulness of the protein should not be ruled out prematurely even though the result obtained here with the rapid test indicated its limitation for case detection with the 42 convalescent phase samples included in this study Nevertheless Gst U274 alone detected a fair proportion 85 of the patient samples tested and further studies of the rapid test using acute phase specimens might prove rewarding Both the recombinant based ELISA and rapid immunochro matographic test developed in this study were found to be highly sensitive and specific with the samples tested When the results were compared the two tests gave an excellent agree ment of 99 6 with a kappa statistic of 1 00 Table 2 and an excellent correlation with an R 2 of 0 988 in relation to their reactivity end points with the seven samples tested Fig 4 This suggested that although the immunochromatographic test was a simple and rapid test that needs no special training to use the performance of the test was fully compatible with that of ELISA This should be a valuable addition to current op tions for combating SARS especially in areas where laboratory facilities are not available In the recent outbreak in Singapore the total number of probable cases of SARS was initially reported as 206 However the number was finally adjusted to 238 after reclassification with a serological test the IFA The confirmation of an addi tional 32 cases suggested the importance of serological tests and the necessity for retesting especially for samples with results initially negative by PCR However although antibody to SARS CoV can be detected in some cases during the acute phase of the disease 1 IgG antibody is believed to reach its peak value around 60 d