CERTIFICATION

AOAC Research Institute

Performance Tested MethodsSM
Certificate No.
051901
The AOAC Research Institute hereby certifies the method known as:

LuciPac A3 Surface
manufactured by
Kikkoman Biochemifa Company
2-1-1, Nishi-shinbashi
Minato-ku, Tokyo 1005-0003
Japan
This method has been evaluated in the AOAC Research Institute Performance Tested MethodsSM Program and found to perform as stated in the applicability of the
method. This certificate indicates an AOAC Research Institute Certification Mark License Agreement has been executed which authorizes the manufacturer to display the
AOAC Research Institute Performance Tested Methods SM certification mark on the above-mentioned method for the period below. Renewal may be granted by the
Expiration Date under the rules stated in the licensing agreement.

Issue Date November 23, 2022

Scott Coates, Senior Director Expiration Date December 31, 2023
Signature for AOAC Research Institute
2275 Research Blvd., Ste. 300, Rockville, Maryland, USA Telephone: +1-301-924-7077 Fax: +1-301-924-7089
Internet e-mail: aoacri@aoac.org * World Wide Web Site: http://www.aoac.org
AUTHORS SUBMITTING COMPANY
ORIGINAL VALIDATION: Natsumi Tanaka, Wataru Saito, and Mikio Kikkoman Biochemifa Company
Bakke 2-1-1, Nishi-shinbashi
MODIFICATION NOVEMBER 2019: Kenta Sakurai and Kazunori Minato-ku, Tokyo 105-0003
Nishimoto Japan

METHOD NAME CATALOG NUMBER

LuciPac A3 Surface 60361

INDEPENDENT LABORATORY AOAC EXPERTS AND PEER REVIEWERS

NSF International Mark Carter1,4, Michael Brodsky2,4, Joseph Odumeru3,4
789 N. Dixboro Rd 1
MC2E, Tennessee, USA
Ann Arbor, MI 48105 2 Brodsky Consultants, Ontario, CANADA
3 University of Guelph, Ontario, CANADA

Q Laboratories 4
Modification November 2019
1930 Radcliff Drive
Cincinnati, OH 45204

APPLICABILITY OF METHOD
Analytes – Adenosine triphosphate (ATP), adenosine diphosphate
(ADP) and adenosine monophosphate (AMP)

Matrixes – Stainless steel

Performance claims – According to the linear regression and other

statistical approaches, the LuciPac A3 Surface for Hygiene Monitoring is
effective at detecting the presence of total adenylate (ATP+ADP+AMP)
on stainless steel surfaces in food processing and food service facilities
with an LOD of 3.3 fmol ATP, 0.9 fmol ADP and 1.8 fmol AMP.

ORIGINAL CERTIFICATION DATE CERTIFICATION RENEWAL RECORD

May 22, 2019 Renewed annually through December 2023.

METHOD MODIFICATION RECORD SUMMARY OF MODIFICATION

1. November 2019 Level 2 1. Addition of Lumitester Smart luminometer.

Under this AOAC Performance Tested MethodsSM License Number, 051901 Under this AOAC Performance Tested MethodsSM License Number, 051901
this method is distributed by: this method is distributed as:
1. AS ONE CORPORATION 1. LuciPac A3 Surface
2. FUJIFILM Wako Pure Chemical Corporation 2. LuciPac A3 Surface
3. KENIS LIMITED 3. LuciPac A3 Surface
4. Nippon Bacterial Test Co., Ltd. 4. LuciPac A3 Surface
5. Weber Scientific 5. LuciPac A3 Surface

PRINCIPLE OF THE METHOD (1)

The principle of detection of A3 is shown in Figure 1. Firefly luciferase can produce light in the presence of ATP, luciferin, oxygen and Mg2+. The amount of light
produced is proportional to the amount of ATP in a sample and therefore ATP can be quantified by measuring the light produced through this reaction using a
luminometer, showing a reading of Relative Light Units (RLUs). This is well known as the ATP method. In order to detect AMP simultaneously and maintain the
light production, ATP was regenerated from AMP using pyruvate orthophosphate dikinase reactions (PPDK) in the presence of phosphoenol pyruvate, inorganic
pyrophosphate (PPi) and Mg2+ (Figure 1). Furthermore, ADP is converted to ATP by pyruvate kinase (PK, Figure 1). This allows the test to detect and quantify total
adenylate and dramatically increases the signal available to the test.
Kikkoman LuciPac A3 Surface, AOAC Performance Tested MethodsSM Certification Number 051901

DISCUSSION OF THE VALIDATION STUDY (1)

ATP tests are commonly used for an assessment of hygienic conditions in food industry. It should be noted that adenylate swabbing assays including ATP and the
A3 test are not for microorganism detection but for cleaning verification because adenylates are not specific to microorganisms as shown in Table 3 and 4.
However, monitoring the surface after cleaning is effective for preventing foodborne illness for the following reasons. First, food residues on surfaces are the
source of nutrients for microorganisms. Second, organic matter can interfere with the antimicrobial activity of disinfectants (5) and decrease sanitation efficiency.
Moreover, cleaning verification also seems to be effective for preventing food allergen cross-contact that can occur via the transfer of allergens in the same facility
or on the same processing line for the allergen-containing and nonallergen-containing foods or ingredients.
A validation study of a conventional ATP monitoring test on stainless steel surfaces has been reported (3). Recently, the LuciPac A3 Surface Hygiene Monitoring
System that can detect ATP+ADP+AMP (A3) has been developed and it shows more advanced sensitivity to determine food/organic debris compared to the
conventional ATP tests (2). However, there is no report about the method validation for A3 assay. Here we report the validation study of the LuciPac A3 Surface
Hygiene Monitoring System under the specific guidelines of the AOAC Research Institute Performance Tested MethodSM program.
Firstly, pure analyte assays were performed to determine the LODs of ATP, ADP and AMP. The results in the method developer laboratory and the independent
laboratory were consistent (Table 2). The LODs were around 10 RLU. According to the regression analyses, LODs can be expressed as ca. 2.5 fmol/assay on a
molecular basis. RSDr values <20% were achieved at or above 2.5 fmol, though RSDr values of analyte-free water and 1.0 fmol adenylate were 20-60% (Table 1).
This study also demonstrated good linearity of detection sensitivity [R2 > 0.9862].
In order to determine the feasibility of detecting food matrix residues on stainless steel surfaces, the surface was treated with dilutions of 5 food matrices, i.e. raw
poultry (raw chicken breast), ready to eat meat product (sliced deli ham), fresh produce and Juice (orange juice), heat processed milk and dairy (yogurt) and
chocolate/bakery products (apple pie). All matrices showed sufficient reactivity as reported previously and a response that varied with dilution (Table 3). Method
Developer Studies demonstrated that pure analyte solutions yielded <20% RSDr (Table 1), but RSDr values of each matrix solution for swabbing assays were <30%.
Independent laboratory Studies demonstrated that RSDr values of each matrix solution for swabbing assays were <26.7% (orange juice) and <42.5% (ham, Table
3). The higher variations of matrixes were likely caused by additional factor, i.e. swabbing technique. Additionally, regarding insoluble food samples, solid and
liquid are separated soon even after careful homogenization. This unavoidable heterogeneity may cause variability in the amount of matrix applied onto the
plates. It should also be considered that all cotton swabs may not be able to pick up the dried solid particles completely. Consistent swabbing technique is
important to minimize the variability. Swabbing an object thoroughly using the entire surface of the swab with rotation is ideal. Ideally the swab should be
slightly bent when exerting appropriate pressing force.
Three pure cultures of microorganisms, a Gram-negative bacterium (C. sakazakii), a Gram-positive bacterium (L. acidophilus), and a yeast species (S. cerevisiae)
were also tested using stainless steel surfaces. As is the case with food matrices, RLU responses to the organism concentration were observed (Table 4). RSDr
values of each microbial solution for swabbing assays (10-35%) were also comparable to the food matrix study. Consequently, validation study using stainless
steel surface demonstrated that the LuciPac A3 Surface Hygiene Monitoring System provides rapid and precise food/organic debris determination.
Disinfectants are used in cleaning to kill microorganisms, and these chemicals may be left on the surface. According to our previous study, sodium hypochlorite
(500 ppm), ethanol (80%) and quaternary ammonium (benzalkonium chloride, 0.1%) inhibit the A3 assays to some extent (ca. 10% inhibition) when 10 µL of
disinfectants were added to the moistened swab (2). In this study, inhibition effects were evaluated using the stainless steel surface model to closely mimic
industrial cleaning practices (Table 5 and 6). Since ethanol can be completely evaporated, another sanitizer for food processing, peracetic acid (6%), was tested
instead in this study. Similar to our previous result, sodium hypochlorite did not affect the result significantly under these conditions. Quaternary ammonium
inhibited 25-30% of the ATP signal. Contrary to our expectations, peracetic acid amplified the RLU output. Acid compounds generally reduce RLU values due to
lowering pH of the reaction mixture from the optimum. The reason of the enhancement by peracetic acid on stainless steel is unclear. The peracetic acid that was
used in this study is composed of hydrogen peroxide, acetic acid, buffer, chelator and stabilizer based on the manufacturer’s information. Peracetic acid (boiling
point: 105°C) and acetic acid (boiling point: 118°C) seem to have been completely evaporated and other components might enhance the measurement values. As
described above, the LuciPac A3 Surface hygiene monitoring system is intended for cleaning verification. Moreover, Table 5 and 6 indicate that it may be affected
by chemical agents. Therefore, the A3 test is recommended to be used after rinsing away sanitizing agents for accurate assessment.
Kikkoman LuciPac A3 Surface, AOAC Performance Tested MethodsSM Certification Number 051901

Table 1. Method developer and independent laboratory pure analyte results using LuciPac A3 Surface/Lumitester
PD-30 system. (A) Adenosine triphosphate (ATP), (B) Adenosine diphosphate (ADP) and (C) Adenosine
monophosphate (AMP) (1)

(A)

(B)

(C)
Kikkoman LuciPac A3 Surface, AOAC Performance Tested MethodsSM Certification Number 051901

Table 2. Estimation of limit of detection (LOD) for adenosine triphosphate (ATP), adenosine diphosphate (ADP), and (C)
adenosine monophosphate (AMP)from the method developer and independent laboratory data of pure analytes using
LuciPac A3 Surface/Lumitester PD-30 system. (1)

a b c Calculated LOD,
Adenylate Xo sb m d e
RLU fmol/assay
ATP 5.2 1.4292 0.0409 10.6 3.3
Method
ADP 4.9 0.3848 0.0955 7.3 0.9
developer
AMP 6.5 0.5710 0.0767 9.6 1.8
ATP 3.1 1.5732 0.0511 9.1 3.0
Independent
ADP 4.0 1.5339 0.0400 9.7 2.9
laboratory
AMP 5.3 0.9548 0.0554 9.3 2.5
a
The mean analytical value of the known negative matrix (Mean RLU for 0 fmol/assay in Table 1).
b
The intercept of the plots of standard deviation vs. mean LuciPac A3 Surface responses (Figure 3).
c
The slope of the plots of standard deviation vs. mean LuciPac A3 Surface responses (Figure 3).
d
Relative Light Unit. Each LOD (RLU) were calcurated using the formula: (X o + 3.3 x s b )/(1–1.65 m )
e
Each LOD (fmol/assay) was calculated by LOD (RLU) using the linearity curves in Figure 2 (Method
developer) and 4 (Independent laboratory).
Kikkoman LuciPac A3 Surface, AOAC Performance Tested MethodsSM Certification Number 051901

Table 3. Replicate Relative Light Unit (RLU), mean RLU, sr and RSDr of the LuciPac A3 Surface method determined with various matrixes (1)

Dilution Replicate RLU Mean RSDr,

Matrix Target RLU s rc
factor 1 2 3 4 5 6 7 8 9 10 RLU %d
1000-500 1000 823 865 919 829 739 790 958 892 655 795 827 89 11
500-200 5000 396 172 192 364 216 222 232 307 293 371 277 81 29
Raw chicken
a 200-75 10000 126 136 144 132 116 112 101 140 86 143 124 19 16
breast
<75 30000 55 43 64 47 62 49 62 79 98 50 61 17 28
Background 14 12 13 21 27 9 26 30 21 25 20 7 37
1000-500 10000 686 533 734 698 710 1163 1075 1098 1018 944 866 218 25
Sliced 500-200 33000 262 182 282 194 294 270 343 380 392 347 295 72 24
200-75 100000 128 93 102 112 129 115 135 128 148 107 120 17 14
deli hama
<75 330000 95 93 58 48 57 58 69 69 57 55 66 16 24
Background 13 15 14 14 12 29e 18 13 15 14 14 2 12
1000-500 5000 556 846 865 672 769 960 986 749 668 617 769 144 19
500-200 10000 193 284 239 241 193 266 208 236 252 324 244 41 17
a
Orange juice 200-75 30000 115 75 84 76 65 85 121 107 90 73 89 19 21
e
<75 100000 25 47 47 54 49 47 36 54 49 46 48 5 11
Background 18 20 25 20 27 29 25 11 13 15 20 6 30
1000-500 2000 857 811 902 940 1004 806 1068 807 980 906 908 90 10
500-200 5000 386 313 306 304 294 468 642 559 523 364 416 124 30
a
Yogurt 200-75 16000 124 119 104 181 86 108 172 106 111 115 123 30 25
<75 32000 43 66 66 55 55 51 76 40 59 47 56 11 20
Background 23 9 18 16 11 17 12 20 18 15 16 4 27
1000-500 300 586 709 647 668 646 714 623 621 631 765 661 54 8
500-200 500 348 424 333 329 376 414 325 314 352 343 356 37 11
Apple piea 200-75 3000 67 89 74 101 107 112 77 81 107 98 91 16 17
<75 5000 31 42 35 41 40 37 31 38 51 48 39 7 17
Background 19 14 18 15 16 14 19 13 23 11 16 4 22
1000-500 60000 785 543 395 465 1011e 461 620 534 571 503 542 113 20.8
500-200 80000 290 200 134 182 279 278 148 411 223 160 231 85 36.9
e
200-75 100000 399 118 173 167 108 186 88 173 105 142 140 36 25.8
Sliced
b 200-75 120000 172 183 403e 281 124 170 101 124 80 90 147 63 42.5
deli ham
200-75 160000 173 304e 114 114 78 70 111 61 57 130 101 38 37.6
<75 400000 153e 50 39 43 45 66 45 48 55 46 49 8 16.3
Background 39 42 34 34 37 42 37 28 40 37 37 4 11.5
1000-500 4000 692 686 516 596 712 721 631 648 661 474 634 83 13.1
500-200 8000 160 219 202 203 239 240 177 208 189 273 211 33 15.7
b 200-75 10000 135 137 144 196 142 195 171 152 148 248 167 36 21.8
Orange juice
200-75 12000 121 244 160 125 225 157 187 215 141 168 174 42 24.3
<75 40000 90 47 55 48 46 46 53 49 56 79 57 15 26.7
Background 41 40 38 40 40 39 31e 40 38 43 40 2 3.9
a
Method developer study.
b
Independent laboratory study.
c
Standard Deviation of Repeatability.
d
Relative Standard Deviation of Repeatability.
e
Excluded from data analysis based on Grubbs' test.
Kikkoman LuciPac A3 Surface, AOAC Performance Tested MethodsSM Certification Number 051901

Table 4. Replicate Relative Light Unit (RLU), mean RLU, sr and RSDr of the LuciPac A3 Surface method determined with various microbes (1)

Theoretical Replicate RLU Mean RSDr,

Organism Target RLU srb
cfu/ml a
1 2 3 4 5 6 7 8 9 10 RLU %c
1000-500 2.0 x 106 730 574 576 675 604 600 644 534 634 536 611 62 10
500-200 8.6 x 105 284 377 293 292 310 278 252 330 329 266 301 37 12
C. sakazaki 200-75 3.0 x 105 146 93 103 108 139 127 108 144 123 139 123 19 15
<75 1.5 x 105 87 65 71 48 34 31 67 86 61 56 61 19 31
Background 0 17 16 35 28 13 14 36 20 19 17 22 8 39
1000-500 2.0 x 105 1258 907 585 660 1081 1086 648 791 674 776 847 227 27
500-200 4.3 x 104 223 230 248 229 320 222 209 254 287 267 249 34 14
L. acidophilus 200-75 2.0 x 104 64 74 56 82 146 53 63 103 113 85 84 29 35
<75 1.0 x 104 34 41 39 64 39 40 44 49 42 52 44 9 19
Background 0 10 9 11 12 31 25 8 9 11 15 14 8 55
1000-500 6.7 x 103 989 1139 818 887 1117 912 926 926 1104 1114 993 116 12
500-200 2.0 x 103 289 298 296 281 226 372 204 256 280 195 270 52 19
S. cerevisiae 200-75 6.7 x 102 143 131 71 152 98 110 67 51 86 80 99 34 35
<75 3.3 x 102 42 31 25 39 33 27 26 18 22 27 29 7 25
Background 0 11 8 33d 11 13 11 17 22 20 13 14 5 33
a
Each value was obtained by deviding the colony forming unit of each undiluted suspention by dilution factors.
The actual amount of organism added to the coupon was 250 μL.
b
Standard Deviation of Repeatability.
c
Relative Standard Deviation of Repeatability.
d
Excluded from data analysis based on Grubbs' test.
Table 5. Replicate Relative Light Unit (RLU) and mean RLU for the effect of common sanitizers on the LuciPac A3 Surface method (1)
Replicate RLU
Water 1000 fmol ATPa 4000 fmol ATP
Sanitizer 1 2 3 4 5 Mean 1 2 3 4 5 Mean 1 2 3 4 5 Mean
None (Water) 23 17 21 15 22 20 147 126 128 160 144 141 417 611 394 589 330 468
Sodium Hypochlorite 31 32 22 30 32 29 148 127 180 168 180 161 382 534 539 506 611 514
Peracetic acid 46 59 83 58 77 65 237 415 208 322 276 292 1239 1343 1235 1352 984 1231
Quaternary ammonium 24 30 28 30 22 27 145 134 110 109 150 130 334 451 282 422 327 363
a
Adenosine triphosphate
Table 6. Effect of common sanitizers on the LuciPac A3 Surface method (1)

Mean RLUa
Water 1000 fmol ATPb 4000 fmol ATP Inhibition, %c
Sanitizer Cd Se CAf SAg CA SA 1000 fmol ATP 4000 fmol ATP
Sodium Hypochlorite 20 31 141 161 468 514 -8 -8
Peracetic acid 20 65 141 292 468 1231 -187 -160
Quaternary ammonium 20 27 141 130 468 363 29 25
a
Relative Light Unit
b
Adenosine triphosphate
c
A negative percent inhibition correlated to an increase in signal. Calculated using mean RLU and the following equation:
Inhibition (%) = {1-[(SA-S)/(CA-C)]}x100.
d
C = Signal from the control (analyte-free water) on the control surface (analyte-free water dried onto the stainless
steel surface).
e
S = Signal from the control (analyte-free water) on the disinfectant surface (disinfectant dried onto the stainless
steel surface).
f
CA = Signal from ATP on the control surface (analyte-free water and ATP dried onto the stainless steel surface).
g
SA = Signal from ATP on the disinfectant surface (disinfectant and ATP dried onto the stainless steel surface).
Kikkoman LuciPac A3 Surface, AOAC Performance Tested MethodsSM Certification Number 051901

DISCUSSION OF THE MODIFICATION APPROVED NOVEMBER 2019 (7)

In the first validation study for the LuciPac A3/the Lumitester PD-30 Hygiene Monitoring System for the detection of ATP, ADP, and AMP from stainless steel
surfaces, pure analyte solutions, detection of food residues and microbial residues on stainless steel surfaces, interference by disinfectants, selectivity of the
method response, instrument variation, lot-to-lot consistency, and accelerated stability were evaluated. In this modification validation study for the new
instruments, Lumitester Smart, pure analyte study and instrument variation were carried out in order to evaluate whether the ability of Lumitester Smart to
detect pure ATP, ADP, and AMP was comparable with that of Lumitester PD-30. Detection of food residues and microbial residues on stainless steel surfaces,
interference by disinfectants, selectivity of the method response, instrument variation, lot-to-lot consistency, and accelerated stability are accordingly ensured by
the previous validation data because these factors depend on the performances of the swab.
The LODs for ATP, ADP, and AMP were 1.6, 3.5, and 3.0 fmol/assay, respectively (Table 2). Pure ATP, ADP, and AMP were detected by the LuciPac A3
Surface/Lumitester Smart system with good linearity (R2 > 0.9866) (Figure 2), and repeatability precision (RSDr: 9.6-18.9 % for 1-100 fmol ATP/assay, 6.4-16.5 % for
2.5-100 fmol ADP/assay, 6.1-15.5 % for 2.5-100 fmol AMP/assay) (Table 1). In our previous report of pure analyte studies using LuciPac A3 Surface/Lumitester PD-
30 system (AOAC Performance Tested MethodSM 051901), the LODs for ATP, ADP, and AMP were 3.0-3.3, 0.9-2.9, 1.8-2.5 fmol/assay, respectively. The
repeatability precision (RSDr) of the measurements were 4.8-16.8 % for 2.5-100 fmol ATP assay, 4.6-23.3 % for 1-100 fmol ADP/assay, and 4.1-24.2 % for 1-100
fmol AMP assay. The linearity (R2) of the measures were 0.9862 or higher.
In the instrument variation studies, no significant difference could be found at any ATP concentration among the three Lumitester Smart (Table 3).
These results indicated that the performance of LuciPac A3 Surface/Lumitester Smart system to detect pure ATP, ADP, and AMP was comparable with that of
LuciPac A3 Surface/Lumitester PD-30 system.

Table 1. Pure analyte results using LuciPac A3 Surface/Lumitester Smart system. (A) Adenosine triphosphate (ATP), (B) Adenosine diphosphate (ADP) and (C)
Adenosine monophosphate (AMP) (7)
A
ATP, fmol/assay
0 1 2.5 5 10 25 100
Mean RLUa 7.6 9.7 14.9 17.1 26.2 53.1 173.6
srb 1.4 1.8 2.5 1.9 3.3 5.2 16.7
RSDr, %c 18.8 18.9 16.9 11.2 12.4 9.9 9.6
Mean fmold -1.1 0.2 3.4 4.7 10.2 26.5 99.6

B
ADP, fmol/assay
0 1 2.5 5 10 25 100
Mean RLU 6.7 10.7 11.9 15.9 25.0 50.7 181.9
sr 2.2 2.5 2.0 2.3 3.3 3.8 11.6
RSDr, % 33.0 22.9 16.5 14.7 13.2 7.5 6.4
Mean fmol -0.5 1.8 2.5 4.8 10.0 24.8 100.1

C
AMP, fmol/assay
0 1 2.5 5 10 25 100
Mean RLU 7.3 10.9 13.2 16.0 25.8 51.0 181.6
sr 2.3 2.9 2.0 1.5 3.3 3.6 11.1
RSDr, % 31.0 26.5 15.5 9.3 12.9 7.0 6.1
Mean fmol -0.5 1.6 2.9 4.5 10.2 24.7 100.1
a
Relative Light Unit. Ten replicates were tested at each concentration.
b Standard Deviation of Repeatability.
c
Relative Standard Deviation of Repeatability.
d Amounts of the adenylate were converted from the mean RLU values using the linearity curves in Figure 2.
Kikkoman LuciPac A3 Surface, AOAC Performance Tested MethodsSM Certification Number 051901

Figure 2. Dose response curves. LuciPac A3 Surface Relative Light Unit (RLU)
responses for (A) adenosine triphosphate (ATP); (B) adenosine diphosphate
(ADP); and (C) adenosine monophosphate (AMP). (7)
A

200
LuciPac A3 Surface RLU

y = 1.6489 x + 9.3693
160 R² = 0.9854

120

80

40

0
0 20 40 60 80 100
ATP (fmol/assay)

200
LuciPac A3 Surface RLU

y = 1.7427 x + 7.5319
160 R² = 0.9931

120

80

40

0
0 20 40 60 80 100
ADP (fmol/assay)

200
LuciPac A3 Surface RLU

y = 1.7335 x + 8.1494
160 R² = 0.9935

120

80

40

0
0 20 40 60 80 100
AMP (fmol/assay)
Kikkoman LuciPac A3 Surface, AOAC Performance Tested MethodsSM Certification Number 051901

Table 2. Estimation of limit of detection (LOD) for adenosine triphosphate (ATP), adenosine diphosphate (ADP), and (C) adenosine monophosphate (AMP)from
the data of pure analytes using LuciPac A3 Surface/Lumitester Smart system. (7)
Calculated LOD,
Adenylate 𝑋𝑋�0a sbb mc
RLUd fmol/assaye
ATP 7.6 0.7497 0.0916 11.9 1.5
ADP 6.7 1.6060 0.0541 13.2 3.2
AMP 7.3 1.5531 0.0517 13.6 3.1
a
The mean analytical value of the known negative matrix (Mean RLU for 0 fmol/assay in Table 1).
b The intercept of the plots of standard deviation vs. mean LuciPac A3 Surface responses (Figure 3).
c The slope of the plots of standard deviation vs. mean LuciPac A3 Surface responses (Figure 3).

� 0 + 3.3 x sb)/(1–1.65 m).

d Relative Light Unit. Each LOD (RLU) were calcurated using the formula: (𝑋𝑋
e Each LOD (fmol/assay) was calculated by LOD (RLU) using the linearity curves in Figure 2.

Table 3. Results of Instrument variation study (7)

Relative Light Unit (RLU)
ATPa, fmol Replicate 23°C 10°C
1d 2d 3d 1d 2d 3d
1 6 8 7 7 6 5
2 6 8 7 8 7 7
3 9 7 7 5 6 8
4 6 5 6 4 6 7
0
5 4 4 5 8 9 8
Mean 6.2 6.4 6.4 6.4 6.8 7.0
srb 1.8 1.8 0.9 1.8 1.3 1.2
RSDr, %c 28.9 28.4 14.0 28.4 19.2 17.5
1 96 96 106 122 110 103
2 99 96 110 113 109 97
3 104 95 90 102 105 100
4 90 93 106 106 112 119
50
5 94 98 95 108 114 115
Mean 96.6 95.6 101.4 110.2 110.0 106.8
sr 5.3 1.8 8.5 7.7 3.4 9.7
RSDr, % 5.5 1.9 8.4 7.0 3.1 9.0
1 978 943 964 1233 931 1203
2 994 958 992 1092 1208 1085
3 941 911 927 1141 1012 1009
4 964 996 887 980 1098 1170
500
5 927 881 878 999 1075 1002
Mean 960.8 937.8 929.6 1089.0 1064.8 1093.8
sr 27.1 44.1 48.9 104.2 103.0 91.4
RSDr, % 2.8 4.7 5.3 9.6 9.7 8.4
a Adenosine triphosphate.
b
Standard Deviation of Repeatability.
c Relative Standard Deviation of Repeatability.
d Serial No. 1: 1911053130070S, 2: 1849053130043S, 3: 1902053130100S.

REFERENCES CITED
1. Tanaka, N., Saito, W., and Bakke, M., Validation Study of LuciPacTM A3 Surface for Hygiene Monitoring through Detection of ATP, ADP, and AMP from Stainless
Steel Surfaces, AOAC Performance Tested MethodsSM certification number 051901. Approved May 2019.
2. Bakke, M., & Suzuki, S. (2018) J. Food Prot. 81, 729-737. doi: 10.4315/0362-028X.JFP-17-432
3. Viator, R., Gray, R. L., Sarver, R., Steiner, B., Mozola, M., & Rice, J. (2017) J. AOAC. Int. 100, 537-547. doi: 10.5740/jaoacint.16-0311
4. Porterfield, R. I., & Capone, J. J. (1984) Med. Devices Diagnostic Ind. 6, 45-50. doi: 10.1007/s11746-001-0401-1.
5. Rutala, W. A., & Weber, D. J. (2016) Am. J. Infect. Control 44, e69-76. doi: 10.1016/j.ajic.2015.10.039
6. Kajiyama, N. & Nakano, E. (1994) Biosci. Biotech. Biochem. 58, 1170-1171. doi: 10.1271/bbb.58.1170
7. Sakurai, K. and Nishimoto, K., Evaluation of Additional Validation Study of New Luminometer for LuciPakTM A3 Surface for Hygiene Monitoring through Detection
of ATP, ADP, and AMP, AOAC Performance Tested MethodsSM certification number 051901. Approved November 2019