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Technical and diagnostic performance of five commercial anti-diphtheria toxoid 1

IgG ELISA Kits 2

Running title: Comparison of anti-diphtheria IgG ELISA kits 3

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Faruq A, Dadson, L., Cox, H., Alcock, F, Parker, A.R.* 5

Binding Site Group Limited, PO Box 11712, Birmingham, B14 4ZB, United 6

Kingdom 7

8

Corresponding author* 9

Binding Site Group Limited 10

PO Box 11712 11

Birmingham 12

B14 4ZB 13

United Kingdom 14

Tel: +44(0)1214361000 15

Fax: +44(0)1214307061 16

Email: [email protected] 17

18

19

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Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00077-10 CVI Accepts, published online ahead of print on 25 August 2010

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Abstract 21

22

The technical and diagnostic performance of five commercially available enzyme-23

linked immunosorbant assays for the measurement of anti-diphtheria toxoid IgG 24

antibodies were evaluated. There was good agreement between the relative 25

sensitivities of the five assays but the relative specificity of one of the assays differed 26

from that of the other four assays. Three of the five assays possessed recoveries of the 27

international reference material NIBSC 00/496 within the range 90%-110% at 28

antibody levels >0.1 IU/mL. The data suggests that there are manufacturer dependent 29

differences in relative sensitivity, specificity and accuracy for the determination of 30

anti-diphtheria toxoid IgG antibodies that could result in different diagnostic 31

interpretations. 32

33

Keywords: Diphtheria toxoid, ELISA, immunodeficiency, immune status34 on January 11, 2021 by guesthttp://cvi.asm

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Introduction. 35

Diphtheria is caused by toxigenic strains of Corynebacterium diphtheriae and 36

is almost entirely preventable through a regular immunisation schedule (9). Since 37

routine immunisation against diphtheria has become standard practice in industrialised 38

nations clinical cases are rare, however it is still a problem in many developing 39

countries such as the new nations formed from the breakup of the USSR (29) as well 40

as in people without adequate immunity. 41

As well as determining rates of immunity within broad populations and the 42

immune status of at risk individuals, accurate measurement of anti-diphtheria toxoid 43

IgG levels are important in assessing the response to vaccination, the efficacy of an 44

immunisation schedule (13, 16, 31), and evaluating individuals for potential 45

immunodeficiency disorders (5). Vaccination response studies are part of the clinical 46

testing recommended for the diagnosis of primary immunodeficiency (2, 5). 47

For determination of anti-diphtheria toxoid IgG antibodies, the in-vivo 48

neutralisation test (NT) and in-vitro Vero cell assay (VCA) are considered the gold 49

standard methods (10, 18). However, ELISA based methodologies offer a simpler, 50

safer technique as live toxin is not required and an ELISA is a more rapid and less 51

expensive technique than a neutralisation test. It is often preferred by clinical 52

laboratories to measure anti-diphtheria toxoid IgG levels. 53

Standardisation of anti-diphtheria toxoid IgG tests has been facilitated by the 54

availability of reference material allowing results to be given in international units 55

(IU). The international standard for anti-diphtheria toxoid IgG immunoglobulin 56

(NIBSC 00/496) was assigned a value of 0.8 IU/mL by comparison to the British 57

standard for Equine anti-diphtheria toxin (NIBSC 66/153) in the mouse in-vivo 58

neutralisation test. The World Health Organisation (WHO) states that a specific IgG 59


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concentration of 0.1 IU/mL is usually considered protective by standard ELISA (27). 60

Many studies have incorporated the WHO guidelines and interpret results as <0.01 61

IU/mL not protective against infection, 0.01-0.09 IU/mL basic protective levels and 62

>0.1 IU/mL as protective levels (4, 5, 7, 8). 63

The titre of anti-diphtheria IgG antibodies in patients with inadequate 64

immunity or an immunodeficiency disease can be very low often <0.1 IU/mL. To be 65

able to accurately and reliably measure low level titres ELISA’s need to be extremely 66

sensitive. This manuscript compares the sensitivity of five commercially available 67

anti-diphtheria toxoid IgG ELISA kits and suggests that the manufacture dependent 68

differences in preparation of these assays may affect the clinical interpretation of data. 69

70

Materials and Methods. 71

Anti-diphtheria toxoid IgG antibodies were measured according to the 72

manufacturers’ instructions using the following ELISA kits with the corresponding 73

measuring ranges: Euroimmun, Lübeck, Germany (0.01-2 IU/mL); Scimedx Corp., 74

New Jersey, USA (0.1-5 IU/mL); Serion-Virion, Würzburg, Germany (0.05-2 75

IU/mL); the Binding Site Group Limited (BS), Birmingham, UK (0.012-3 IU/mL) and 76

Genzyme Virotech, Rüsselsheim, Germany (0.1-5 IU/mL). The time taken to run the 77

assays were as follows: Euroimmun, 105 mins; Scimedx Corp., 90 mins; Serion-78

Virion, 120 mins; BS, 90 mins; and Genzyme Virotech, 90 mins. Results were 79

generated as per the manufacturer’s instructions. Assays were considered valid when 80

quality control parameters were in range as per the manufacturer’s product insert. 81

Intra-assay precision for all five kits was measured using three serum samples 82

(low, medium and high level) and assayed in six well repeats at the same time. For 83

Euroimmun, Serion and BS a further sample was used for measurement of precision 84


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<0.1 IU/mL. For inter-assay precision, the same measurements were performed over 85

two consecutive days. The intra- and inter-assay precision was assessed by calculating 86

the coefficient of variation. 87

Normal human sera (unknown vaccination status) and pre- and post-diphtheria 88

toxoid vaccination serum samples were obtained from Research Sample Bank, Inc., 89

Florida, USA and Golden West Biologicals Inc., California, USA and stored at -20oC 90

prior to testing. 91

For the comparison between two assays we calculated the relative sensitivities 92

and relative specificities on the same set of samples run in both assays. The 93

determination of whether a sample was positive or negative was relative to the cut off 94

value of 0.1 IU/mL in a particular assay. The values obtained served only for the 95

comparison between the two assays and the results were expressed as a percentage. 96

To calculate: relative sensitivities: ([number of samples positive in assays A and 97

B]/{[number of samples positive in assays A and B]+[number of samples positive in 98

assay A and negative in assay B]} *100); relative specificities: ([number of samples 99

negative in assays A and B]/{[number of samples negative in assays A and 100

B]+[number of samples positive in assay A and negative in assay B]} *100). 101

The reference material NIBSC 00/496 (NIBSC, Hertfordshire, UK) was used 102

to evaluate the calibration in the ELISA assays. For use, NIBSC 00/496 was 103

reconstituted according to the manufacturer’s instructions (working concentration of 104

1.6 IU/mL) and serially diluted with the appropriate sample diluents to a final 105

concentration of 0.012 IU/mL. Calibration of each assay was assessed by calculating 106

the “recovery” of NIBSC 00/496 by obtaining values (IU/mL) for serially diluted 107

NIBSC 00/496 from the assay calibration curve and comparing them to the expected 108


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values according to this equation ([obtained NIBSC value/expected NIBSC 109

value]*100). The results are expressed as a percentage. 110

Response to diphtheria vaccination was assessed using twelve pre- and post-111

vaccination sample pairs, assayed over two consecutive days. The samples were 112

evaluated for a significant fold increase in titres between pre-vaccination titre and 113

post-vaccination titre. A significant increase has been defined in literature as 4-fold 114

(31). 115

For statistical analysis of the data Wilcoxon’s rank sum test and Freidman’s 116

analysis were applied using the Analyse-it Software Ltd, UK. A p value <0.05 was 117

considered significant. 118

119

Results. 120

Intra- and inter-assay precision were calculated for each kit (Table 1). Three 121

random samples were selected that could be read from all five calibration curves in 122

the measuring range of 0.1-2 IU/mL. The intra and inter-assay precision for the three 123

samples ranged from 3.4%-17.4% and 4.6%-27.3% respectively with the lowest 124

values achieved with Scimedx (4.6%-5.8% and 4.6%-7.5%) and BS (3.4%-3.8% and 125

5.8%-6.7%) assays. Poor inter-assay precision (>15%) was evident for the low sample 126

on the Euroimmun assay and the high sample on both the Euroimmun and Serion 127

assays. To further challenge precision at the bottom end of the curve on the three 128

assays with measuring ranges lower than 0.1 IU/mL (Serion 0.05 IU/mL; BS 0.01 129

IU/mL; and Euroimmun 0.01 IU/mL) precision with another sample was assessed. 130

This sample was randomly selected similar to the three samples used above since it 131

gave a measureable value <0.1 IU/mL in all three assays. The mean precision of the 132


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sample in the Serion assay was 0.052 IU/mL (CV=15.2%), BS 0.09 IU/mL 133

(CV=4.2%) and Euroimmun 0.031 IU/mL (CV=10.3%). 134

In order to assess the performance of the five ELISA assays, levels of 135

antibodies to diphtheria toxoid in fifty six serum samples were determined and 136

quantified (Figure 1). On the Scimedx, Serion and Virotech assays not all samples 137

could be measured due to the limitation of the lower end of the measuring range and 138

were therefore not included in the figure. All five ELISA assays identified a number 139

of samples with titres of anti-diphtheria IgG antibodies below the WHO 140

recommended level of protection (0.1 IU/mL); Euroimmun (37/56 samples; 66%), 141

Scimedx assay (3/56 samples; 5.3%), Serion assay (21/56 samples; 38%), BS (26/56 142

samples; 46.4%) and Virotech assays (9/56 samples; 17%). 143

Figure 1 also shows there were no statistical differences between the 144

Euroimmun and BS assays (Wilcoxon test, p=0.375) and the Scimedx, Serion and 145

Virotech assays (Friedman’s test, p=0.295) but there was a significant difference 146

between the two groups of data (p<0.0001 Wilcoxon test). The figure also suggests 147

that there was not an even distribution of sample values around the median in the 148

Euroimmun and Scimedx assays. Using the samples that were quantifiable in all five 149

assays, there was good agreement with regards positivity (>0.1 IU/mL) and negativity 150

(<0.1 IU/mL) between the five assays. A comparison of relative sensitivity between 151

the five assays was 88.6% - 100% and relative specificity between four of the assays 152

was 72.7%-90.5% with the exception of the Scimedx assay (45% - 52.6%). 153

To assess the accuracy of calibration, the recoveries of serially diluted NIBSC 154

00/496 reference material with known titres, read from the respective calibration 155

material were assessed and expressed as a percentage target value (Table 2). To allow 156

fair comparison across all five assays, recovery across a measuring range of 0.11-3 157


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IU/mL was compared. Two of the five assays (BS and Virotech) gave consistent and 158

accurate recoveries of the reference material across the range tested with %CV <10%. 159

When recoveries around the protective cut off of 0.1 IU/mL were assessed, only 160

Scimedx (95.1%), BS (97.1%) and Virotech (91.5%) showed recoveries within 10% 161

of the target value. When the diluted reference material at target values of 0.037 and 162

0.012 IU/mL, were run on the two assays designed to measure that low, the values 163

were 108.1% and 130.3% respectively in the BS assay but were undetectable in the 164

Euroimmun assay. 165

Levels of anti-diphtheria toxin IgG antibodies were determined in twelve 166

paired pre- and post-vaccination samples (Table 3). It was not possible to obtain 167

quantitative values for all pre-vaccination samples by all five assays as some samples 168

were below the limit of detection in certain assays. 169

It has been proposed that a four-fold, or greater, increase in the specific 170

antibody titre is a clinically significant response to diphtheria vaccine (16). It has also 171

been suggested that a ‘non responder’ would exhibit a less than 2.6-fold increase in 172

antibody titres after vaccination or antibody titres less than the protective titre post-173

vaccination (17, 27). The titres of anti-diphtheria IgG antibodies were <0.1 IU/mL for 174

the pre-vaccination samples 9 and 21 in the Euroimmun assay and for pre-vaccination 175

samples 10, 14, 17, 19, and 26 were >0.1 IU/mL in the Scimedx assay which were out 176

of consensus with titres measured in the other four assays. In the Scimedx assay, anti-177

diphtheria IgG titres recorded in samples 10, 16 and 19, a four-fold increase in titre 178

was not achieved post-vaccination but the pre-vaccination levels indicated titres >0.1 179

IU/mL. 180

For both the Serion and Virotech assays it was difficult to accurately measure 181

an increase in titre for samples 10, 14, 17, 19 and sample 22 in the Serion assay and 182


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sample 26 in the Virotech assay as the pre-vaccination anti-diphtheria IgG antibody 183

titres were lower than the measuring range of the kit. Four fold increases in antibody 184

titre could not be measured in either assay for sample 16 however the pre-vaccination 185

level >0.1 IU/mL and no four fold increase in titre was measured in sample 26 in the 186

Serion assay but the post-vaccination level was >0.1 IU/mL. The value measured for 187

pre-vaccination sample 22 in Virotech assay was >0.1 IU/mL which was out of 188

consensus with values obtained in the other four assays which either recorded values 189

lower than the measuring range or falling in the range but <0.1 IU/mL. 190

In the BS assay a four fold increase in anti-diphtheria IgG titres were not 191

obtained for samples 15, 16 and 26 but in the two former samples the pre-vaccination 192

titre was >0.1 IU/mL and in sample 26 the post-vaccination tire was >0.1 IU/mL. In 193

all five assays a four fold increase in titre could not be measured for samples 20 and 194

28. However, for sample 20 all pre-vaccination levels >0.1 IU/mL and both pre and 195

post-vaccination titres <0.1 IU/mL for sample 28. 196

197

Discussion. 198

Although reports have suggested the anti-diphtheria IgG antibody titre 199

required for adequate protection is >0.1 IU/mL (27), several reports have suggested 200

that sensitive quantitation of specific IgG levels between 0.01-0.1 IU/mL are also 201

important. Antibody levels within this range provide minimal protection and 202

quantification may provide important clinical information. 203

There is debate as to whether in-vitro techniques for measuring anti-diphtheria 204

IgG antibodies are as sensitive as the in-vivo neutralisation tests. It has been 205

suggested that inaccuracies with ELISA may be due to the detection of non-206

neutralising antibodies directed towards an immunogenic but non-toxic part of the 207

toxoid (22, 23). In these studies it is not stated whether these ELISA assays were 208


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calibrated to any standard such as NIBSC 00/496. When using standardised in-vitro 209

ELISA for the measurement of anti-diphtheria IgG antibodies the correlation of 210

sensitivities with in-vivo neutralisation assay and in-vitro tissue culture method are 211

good as low as 0.01 IU/mL (17, 30). At titres <0.01 IU/mL however the correlation 212

has been shown to be poor (14, 17). 213

In the present study we have compared the assay performance of five different 214

commercially available anti-diphtheria toxoid IgG ELISA assays. The data suggests 215

that considerable variation exists between the five assays. The range of %CV for both 216

intra-assays and inter-assays were 3.4%-17.4% and 4.6%-27.3% respectively with 217

samples of antibody titres >0.1 IU/mL and 4.2-15.2% for samples <0.1 IU/mL. The 218

serum sample data readings suggests that the Euroimmun and BS assays give similar 219

results but that they are significantly different to the Scimedx, Serion and Virotech 220

results. The sensitivities between the five assays were comparable but the Scimedx 221

assay had lower specificity with respects to the other four assays. 222

Comparison of the accuracy of calibration to the international reference 223

standard NIBSC 00/496 revealed interesting results. Only three of the assays 224

possessed <15% mean variation between the assays calibrator material and reference 225

standard at anti-diphtheria IgG antibody levels >0.1 IU/mL. The other two assays 226

showed large mean variation of 25.4-44.3%. At levels <0.1 IU/mL, one assay had a 227

mean variation of 19% and the other failed to detect the low level anti-diphtheria IgG 228

antibodies. 229

Analysis of the anti-diphtheria IgG levels in pre- and post-vaccination serum 230

further highlighted variation. Depending on the assay chosen a sample could be 231

interpreted as possessing a protective or non protective level of anti-diphtheria IgG 232

antibodies and accurate assessment of fold increase in titres could be difficult. 233


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A recent independent study has also highlighted the differences that exist 234

between commercial anti-diphtheria IgG ELISA kits. Di Giovine et al (6) assessed the 235

performance of five commercial anti-diphtheria IgG ELISA assays using samples 236

with values determined in the gold standard VCA. The correlation coefficients 237

between the individual assays and the VCA ranged from 0.6 to 0.85 and in three of 238

the assays the majority of VCA positive samples were <0.1 IU/mL. The data clearly 239

demonstrated that manufacture dependent differences between anti-diphtheria IgG 240

ELISA assays exist. 241

Since all assays were performed by the same technician and the protocols for 242

all commercial assays were similar such variations that affect antibody:antigen 243

interaction may arise at several points in assay manufacture. The variation in precision 244

data suggests possible differences in antigen source, preparation or plate coating 245

techniques and the difference in the data sets imply that different types of blocking 246

agents may have been utilised or that plate blocking may have occurred to different 247

extents. Some of the variation is undoubtedly due to differences in the setting of the 248

calibration and it is not disclosed by all manufacturers how the calibration has been 249

set with regards to the use of an international standard. Potentially also, the types of 250

buffer systems used in the assays could also influence the antibody:antigen 251

interaction. Almost certainly the data suggests that there is a need for some level of 252

agreed standardisation in the preparation of these assays. 253

The sensitive measurements of anti-diphtheria IgG antibodies are important 254

clinically for two reasons: assessment of immune status and measurement of immune 255

response. The WHO have suggested the level of anti-diphtheria toxoid IgG antibodies 256

required for optimal protection from diphtheria infection is >0.1 IU/mL. However, the 257

levels of circulating anti-diphtheria IgG antibodies required for basic immunity in 258


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man has been estimated to be 0.01 IU/mL by the Schick skin test (12, 24, 25) and 259

many studies have reported that levels in the general population are <0.1 IU/mL and 260

in some cases <0.01 IU/mL (1, 3, 11, 19, 20, 21, 26, 28, 32). Maple and colleagues 261

showed that from the age of 14 years up to <54 years of age the percentage of the 262

population (n=3088) with anti-diphtheria toxoid IgG levels >0.1 IU/mL decreased 263

from ~40% to ~5%. Within the same groups the percentage of the population with 264

levels <0.01 IU/mL increased from ~10% to ~40% (15). A study by Edmonds and 265

colleagues found similar results (7). In the present study the standard deviation of the 266

mean titre in fifty six random serum samples and in the pre-vaccination samples 267

suggested that a considerable percentage of samples have anti-diphtheria IgG 268

antibody titres <0.1 IU/mL (between 5.3% and 66% depending on the assay used). 269

The response of the immune system to the challenge of a vaccine can aid the 270

immunologist in their diagnosis of an immunodeficiency (5). Studies have suggested 271

that responders, with an efficient immune response, produce a 4-31.1 fold increase in 272

specific antibody titre to diphtheria vaccine (2, 16, 26) whereas non-responders, 273

possessing a defective immune response, only show an approximate 2.6 fold increase 274

in titre (16) or an antibody titre post-vaccination of <0.1 IU/ml (27). Furthermore, two 275

studies have suggested that the percentage of non responders to the diphtheria vaccine 276

could be between 4% and 14% depending on the age group and vaccination regime 277

(11, 20). 278

In conclusion, we have provided a study to evaluate the performance by five 279

different commercial ELISA assays in the measurement of anti-diphtheria toxoid IgG 280

levels. Given the clinical use of the information derived from the test it is essential 281

that sensitive and accurate ELISA assays are utilised. We conclude that among the 282

commercially anti-diphtheria IgG ELISA assays tested in this manuscript there are 283


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significant differences that exist with regards to precision, relative sensitivity, relative 284

specificity and calibration. Further studies are required to understand the clinical 285

implications that arise from the use of different ELISA assays with differing levels of 286

sensitivity and these significant manufacture dependent differences. 287

288

Acknowledgements. 289

290

We thank Dr. Richard Hughes for his useful comments in the preparation of this 291

manuscript.292

All authors are employees of the Binding Site Group Ltd, which produces one of the

assays used in this study.


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Figure Legend

Figure 1. Box plot of normal samples with quantitative values assayed in the five

ELISA assays (Euroimmun, n=52; BS, n=56; Serion, n=40; Scimedx, n=53;

Virotech, n=47).


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Table 1. Intra- and inter-precision calculated in five commercial ELISA assays.

Euroimmun Scimedx Serion BS Virotech

Serum Sample Intra Inter Intra Inter Intra Inter Intra Inter Intra Inter

Mean (IU/mL) 0.44 0.44 0.65 0.65 0.48 0.48 0.52 0.52 0.52 0.52

SD 0.06 0.12 0.03 0.03 0.03 0.03 0.02 0.03 0.03 0.06 Low

CV % 13.6 27.3 4.6 4.6 6.3 6.3 3.8 5.8 5.8 11.5

Mean (IU/mL) 0.96 0.96 2.92 2.92 1.50 1.50 0.89 0.89 1.36 1.36

SD 0.10 0.14 0.17 0.22 0.11 0.12 0.03 0.06 0.06 0.09 Medium

CV % 10.4 14.6 5.8 7.5 7.3 8.0 3.4 6.7 4.4 6.6

Mean (IU/mL) 0.46 0.46 0.96 0.96 0.45 0.45 1.04 1.04 0.81 0.81

SD 0.08 0.09 0.05 0.07 0.03 0.07 0.04 0.06 0.05 0.06 High

CV % 17.4 19.6 5.2 7.3 6.7 15.6 3.8 5.8 6.2 7.4


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Table 2. Mean percentage recovery from three repeat assays of NIBSC 00/496 standard in five commercial ELISAs.

Recovery (%) Target

(IU/mL) Euroimmun Scimedxa Serion

b BS Virotech

a

3.00 45.5 149.3 NA 85.3 107.6

1.00 98.0 127.7 104.9 89.7 114.1

0.33 83.6 86.1 94.5 94.8 100.2

0.11

37.5

95.1

82.3

97.1

91.5

0.037 0.00 N/A N/A 108.1 N/A

0.012 0.00 N/A N/A 130.3 N/A

Meanc 66.1 114.7 93.9 91.7 103.3

CV 44.3 25.4 12.1 5.8 9.4

NA= not applicable.

a Measuring range of these kits were 0.1-5 IU/mL (see methods and materials).

b Measuring range of this kit was 0.05-2.0 IU/mL (see methods and materials).

c Mean value and CV are for the recovery of samples 0.11-3.00 IU/mL

To calculate, values (IU/mL) for serially diluted NIBSC 00/496 were obtained from a calibration curve and compared to the expected value

according to this equation ([obtained NIBSC value/ expected NIBSC value]*100).


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Table 3. Pre and post vaccination data from five commercial ELISA tests.

The samples were assayed in duplicate wells at the same time in three separate assays. All assays were valid and the data for the pre- and post-

vaccination samples represent the mean value derived from the three independent assays.

EUROIMMUN SCIMEDX SERION BS VIROTECH

Sample ID

Pre

Post

Fold

Pre

Post

Fold

Pre

Post

Fold

Pre

Post

Fold

Pre

Post

Fold

9

0.093

1.089

11.7

0.186

1.547

8.3

0.113

1.286

11.4

0.129

0.914

7.1

0.129

1.351

10.4

10 0.018 0.174 9.7 0.103 0.333 3.2 <0.05 0.310 - 0.046 0.218 4.8 <0.1 0.246 -

14 0.018 0.822 46.3 0.103 1.257 12.2 <0.05 0.861 - 0.049 0.838 17.1 <0.1 0.891 -

15 0.232 1.271 5.5 0.246 2.445 10.0 0.458 >2.000 - 0.431 1.458 3.4 0.183 1.826 10.0

16 0.077 0.494 6.4 0.175 0.560 3.2 0.220 0.608 2.8 0.191 0.417 2.2 0.251 0.722 2.9

17 0.045 0.884 19.4 0.138 1.186 8.6 <0.05 0.776 - 0.065 0.567 8.8 <0.1 0.709 -

19 0.011 0.146 13.0 0.110 0.228 2.1 <0.05 0.278 - 0.044 0.266 6.0 <0.1 0.211 -

20 0.160 0.495 3.1 0.622 1.034 1.7 0.630 0.930 1.5 0.377 0.614 1.6 0.339 0.620 1.8

21 0.069 1.233 17.8 0.137 3.806 27.7 0.265 >2.000 - 0.216 1.476 6.8 0.164 1.744 10.7

22 <0.01 0.568 - <0.1 0.907 - <0.05 0.557 - 0.025 0.460 18.2 0.154 0.765 5.0

26 0.054 0.308 5.7 0.149 0.336 2.3 0.061 0.233 3.8 0.065 0.178 2.7 <0.1 0.252 -

28 0.014 0.018 1.3 <0.1 <0.1 - <0.05 <0.05 - 0.021 0.035 1.7 <0.1 <0.1 -

Mean

0.072

0.625

0.197

1.240

0.291

0.585

0.138

0.620

0.203

0.849

SD 0.069 0.431 0.156 1.068 0.216 0.387 0.140 0.473 0.078 0.569

SEM1 0.020 0.125

0.045 0.308

0.062 0.112

0.040 0.137

0.023 0.164

1 SEM is the standard error of the mean for all readable pre- or post-vaccination samples.


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Figure 1

EUROIM

MUN

BS

SERIO

N

SCIM

EDX

VIR

OTECH

0.001

0.01

0.1

1

10p=0.375p=0.375p=0.375

p=0.295

p<0.0001

IU/m

l


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