Long-term storage stability of multiplex immunochromatographic assay strips for serologic monitoring of infectious diseases in laboratory rodents

Abstract

Serological monitoring of infectious diseases in laboratory rodents is vital for ensuring animal health. Therefore, we have previously developed a multiplex immunochromatographic assay (ICA) to detect antibodies against six pathogens as follows: mouse hepatitis virus, Sendai virus, and Clostridium piliforme (Tyzzer’s disease) in mice, and Seoul type orthohantavirus, Sendai virus, and sialodacryoadenitis virus in rats. However, the preparation of these strips is time-consuming and labor-intensive. Therefore, this study aimed to determine the long-term storage conditions for the developed multiplex ICA strips. The multiplex ICA strips stored in the polyethylene zipper-sealed bags (poly_bags) at −30°C maintained detectable reactivity for all six targeted pathogens for 36 months, regardless of the presence or absence of desiccants, oxygen absorbers, or a vacuum state. However, when stored at 18–28°C (room temperature) and 4°C, the detectable reactivity of the multiplex ICA strips stored in poly_bags was reduced for some of the six targets pathogens at 18 months after storage. This reduction was not prevented by the storage with the desiccants and in the vacuum-sealed poly_bags (vacuum_bags). The oxygen absorber reduced detectability of the multiplex ICA strips. These results indicate that storing ICA strips in poly_bags at −30°C, which is a simple method, is able to maintain detectability long-term for the strips format and pathogen targets examined in this study.

INTRODUCTION

Microbiological monitoring of laboratory animals used in experimental studies is essential from the viewpoints of research reliability, reproducibility, and animal welfare (3Rs). The microbiological status of laboratory rodents is typically monitored by exposing sentinel animals to potentially contaminated soiled bedding or exhaust air from individually ventilated cages for several weeks. Enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), culture, and microscopic examination are the main tests used for the serological diagnosis of infectious diseases and the detection of pathogens in laboratory rats and mice. Commercially available ELISA kits, such as MONILISA^® (Wakamoto Pharmaceutical, Tokyo, Japan), are mainly used for in-house monitoring.

Assessment of microbial status without euthanizing and distressing test animals, including sentinels, is now considered important from the viewpoint of the 3Rs. Multiplex microbead immunoassays and PCR-based detection of antigens in feces have been reported as methods for diagnosing infectious diseases without euthanizing or distressing animals [5, 11,12,13, 15, 16]. The multiplex immunochromatographic assay (ICA) is a rapid and simple serologic test specifically designed for the detection of pathogens from a small amount of blood, thereby reducing euthanasia rates and distress in test animals. The developed multiplex ICA was used to detect mouse hepatitis virus (MHV), Sendai virus (previously known as hemagglutinating virus of Japan) (HVJ), and Clostridium piliforme, which is the pathogen causing Tyzzer’s disease (Tyzzer) in laboratory mice and HVJ, Seoul type orthohantavirus (Hanta), and sialodacryoadenitis virus (SDAV) in laboratory rats [18, 19].

When an infectious disease outbreak is confirmed, rapid screening tests must be conducted to understand the extent of its spread. Therefore, the long-term storage of the developed multiplex ICA strips is important for implementing rapid response measures during an infectious outbreak, as the preparation of the strips is time-consuming and labor-intensive. The strips of the developed multiplex ICA were stored at 18–28°C (room temperature) in a dry box until use and was within 3 months [18, 19]. However, for storage of more than 3 months of the developed multiplex ICA strips, the effects of storage conditions such as temperature and humidity on detection sensitivity and specificity of antibody and storage period are unknown. Moreover, there have been also few reports on the storage conditions and stability of other ICA strips. In this study, we aim to determine suitable long-term storage conditions for the developed multiplex ICA strips for simultaneous serodetection of MHV, HVJ, and Tyzzer in mice, and Hanta, HVJ, and SDAV in rats.

MATERIALS AND METHODS

Control antigen

Inactivated fractions of purified MHV (S strain) and HVJ (MN strain) virions and a purified and inactivated Clostridium piliforme (RJ strain), which is the pathogen causing Tyzzer, were provided by the ICLAS Monitoring Center (Central Institute for Experimental Medicine and Life Science Central, Japan) [6, 8]. A recombinant antigen for Hanta diagnosis was prepared as previously described [1, 2, 23].

Serum

Serum samples positive for antibodies against MHV, SDAV, HVJ and Tyzzer were provided by the ICLAS Monitoring Center. These samples were diagnosed through ELISA and an immunofluorescent antibody assay as previously described [8]. The protocol for collecting positive serum samples was approved by the IACUC according to the Regulations for Central Institute for Experimental Medicine and Life Science Central, Japan. The regulations were established in compliance with the law for humane treatment and management of animals.

Serum samples positive for antibodies against Hanta were diagnosed by using ELISA and an immunofluorescent antibody assay as previously described [1, 2].

Lot numbers of the serum samples positive for antibodies against each antigen were as follows: mouse MHV No.1, mouse HVJ No.2, mouse Tyzzer No.9, rat SDAV No.5, rats HVJ No.5, and anti-Seoul virus strain Gou3 (Hanta). To prevent deterioration caused by freezing and thawing, the serum samples were stored at −80°C in amounts required for one analysis.

Multiplex ICA

The multiplex ICA strips for detection of serum antibodies to MHV, HVJ, and Tyzzer in mice and SDAV, HVJ, and Hanta in rats were prepared as described previously [18, 19].

(i) Preparation of a colloidal gold conjugate: A gold colloid solution (WRGH2, Wine Red Chemical, Tokyo, Japan) was mixed with an equal volume of 10 mM Tris-HCl (pH 8.15). Rabbit anti-rat IgG antibody (Sigma, St. Louis, MO, USA) and protein A (Nacalai tesque, Kyoto, Japan) solution were diluted to 1.5 mg/mL by using 10 mM Tris-HCl (pH 8.15) and then mixed with 2 volumes of the colloidal gold dilution in a siliconized tube. After brief sonication, the mixture was incubated for 20 min at room temperature, after which a 1/3 volume of blocking solution (2.5% casein sodium (Wako, Osaka, Japan) in 10 mM Tris-HCl (pH 9.2)) was added. The mixture was sonicated briefly, incubated for 20 min at room temperature, and centrifuged at 14,000 × g for 15 min. After removal of the supernatant, the pellet was resuspended in blocking solution to the initial volume of colloidal gold. After brief sonication, the mixture was incubated for 20 min at room temperature and then centrifuged at 14,000 × g for 15 min. Finally, the supernatant was removed, and the pellet was resuspended with 200 μL of blocking solution.

(ii) Preparation of a conjugate pad: A glass-fiber conjugate pad (GFDX103000; MilliporeSigma, Billerica, MA, USA) was soaked in 0.5% casein in 20 mM sodium phosphate buffer (pH 7.8) for 20 min. The pad was washed with 3.0% sucrose in double-distilled water and air-dried overnight. The pad was coated with the prepared colloidal gold conjugate at a dose of 200 μL/30 cm. The pad was air-dried overnight and stored at room temperature.

(iii) Preparation of a multiplex ICA strip: For diagnosis of MHV, HVJ and Tyzzer in mice, inactivated fractions of purified MHV (S strain) and HVJ (MN strain) virions and a purified and inactivated Clostridium piliforme (RJ strain) were used respectively. For diagnosis of SDAV, HVJ and Hanta in rats, inactivated fractions of purified MHV (S strain), which cross-reacts with SDAV [10], and HVJ (MN strain) virions and the recombinant antigen of Hanta were used respectively. Each antigen for test line was diluted with 0.05% casein sodium in 10 mM PB (pH 7.2) to adjust the concentrations to 1.0 mg/mL for MHV and Hanta antigens, to 0.5 mg/mL for HVJ and Tyzzer antigens. Rabbit IgG (FUJIFILM Wako Pure Chemical, Osaka, Japan) for control line of strip for mice, and anti-rabbit IgG antibody (MP Biomedicals, Solon, OH, USA) for control line of strip for rats, were diluted with 0.05% casein sodium in 10 mM PB (pH 7.2) to adjust the concentrations to 0.15 mg/mL. These reagents were immobilized on a nitrocellulose membrane (60 mm × 300 mm) (Hi-Flow Plus 135 Membrane Card; MilliporeSigma) by using a pen (Super brush for Copic Sketch and Ciao; Too Marker Products, Tokyo, Japan) to draw thin lines for the reagents at the test line position (each antigen) and control line position (anti-rabbit IgG antibody for rats or rabbit IgG for mice).

An ICA strip containing the 3 antigens and the control as separate lines was used as a multiplex ICA strip. After the membrane had been dried at room temperature for 15 min, it was soaked in 20 mM sodium phosphate buffer containing 0.5% casein (pH 7.8) for 20 min to block the unsaturated area. Then the membrane was washed twice (5 min each time) with double-distilled water, soaked in 3.0% sucrose in double-distilled water, and air-dried overnight. Finally, a sample pad (20 mm × 300 mm) (Chromatography Paper; Whatman, GE Healthcare BioSciences, Pittsburgh, PA, USA), conjugate pad, nitrocellulose membrane and absorbent pad (50 mm × 300 mm) (catalog no. CFSP20300, Cellulose fiber sample pad; MilliporeSigma, Billerica, MA, USA) were assembled on a membrane card (MilliporeSigma). The combined membranes were cut into 2-mm-wide strips by using a paper cutter. The structure of the ICA strip is shown in Fig. 1A.

Fig. 1.

Schematic diagram demonstrating the detection method and presentation of a multiplex immunochromatographic assay (ICA) strip. A) Structure of the multiplex ICA. Antigens were placed at the test line. Rabbit IgG and anti-rabbit IgG antibodies were placed at the control line for mice and rats, respectively. The ICA strip consisted of four membrane pads: sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad. The conjugate pad contained a protein A-colloidal gold conjugate used for serologic diagnosis of infectious diseases in mice or an anti-rat IgG antibody colloidal gold conjugate, which is used for the serologic diagnosis of infectious diseases in rats. B) Detection method. Serum (0.75 μL) was diluted with 150 μL of phosphate-buffered saline (PBS) and placed in a microtube. After the strip was dipped in the solution for 15 min, the test line (yellow arrow) and control line (red arrow) were examined visually. A sample in which only the control line appeared was regarded as negative (1), whereas a sample in which both the test and control lines appeared was regarded as positive (2).

(iv) Procedure for the multiplex ICA: A 0.75 mL of the serum sample positive for antibodies against each antigen was added to 150 mL of phosphate-buffered saline (PBS), making a 1:200 dilution. An ICA strip was put directly into a microtube containing PBS-diluted serum (Fig. 1B). After the tube had remained still for 15 min, the ICA strip was examined visually. The detectability of the ICA strips was determined visually by all co-authors. When the intensity of both the control and test lines was clear, it was regarded that detectability of the ICA strip was maintained. The following cases were regarded that detectability of the ICA strip was not maintained: 1) the intensity of both the control and test lines in each positive serum were reduced, making evaluation difficult, 2) neither the control nor test lines against each positive serum was detected, 3) the control line was detected but the test line was not detected, 4) the test line was detected but the control line was not detected. When bands were detected on the test lines of antigens other than the target antigen, it was judged as nonspecific reactions.

Storage of multiplex ICA strips

As shown in Fig. 2A, the multiplex ICA strips were stored in polyethylene zipper-sealed bags (poly_bags) (140 × 100 × 0.08 mm, cold resistance temperature: −30°C) (Unipack E-8; SEISANNIPPONSHA, Tokyo, Japan) and in vacuum-sealed poly_bags (vacuum_bags) at 18–28°C (room temperature), 4°C, and −30°C in dark for 6, 18, and 36 months. The vacuum state was achieved by removing the air using a vacuum sealer (Vacuum Sealer Economy VS-400S; AS ONE, Osaka, Japan). Moreover, the following conditions were set for each storage condition: 1) stored without both desiccants (Synthetic Zeolite Plate Strong Desiccant AZ10G-100; AS ONE, Osaka, Japan) and oxygen absorbers (Oxygen absorber A-750HS; AS ONE), 2) stored with desiccants only, 3) stored with oxygen absorbers only, 4) stored with both desiccants and oxygen absorbers (Fig. 2B). The poly_bags used for storage at −30°C was ensured that its airtightness was maintained after being brought back to room temperature for analysis.

Fig. 2.

Storage conditions of the multiplex immunochromatographic assay (ICA) strip. A) Conditions for the airtightness of storage bags of the multiplex ICA strips: (1) polyethylene zipper-sealed bags (poly_bags), (2) vacuum-sealed poly_bags (vacuum_bags). The vacuum state was achieved by removing the air using a vacuum sealer. B) Conditions of desiccants and oxygen absorbers in poly and vacuum_bags: (1) without both desiccants and oxygen absorbers, (2) with desiccants only, (3) with oxygen absorbers only, (4) with both desiccants and oxygen absorbers.

RESULTS

Evaluation of strip reactivity of the multiplex ICA strips for MHV, HVJ, and Tyzzer detection in mice under different storage conditions

Multiplex ICA strips stored at room temperature in poly_bags without both desiccants and oxygen absorbers were evaluated for detectability over time (Table 1). It was regarded that the detectability of Tyzzer and the control lines were maintained for up to 6 months after storage, whereas the detectability of the HVJ, MHV, and control lines were maintained for up to 18 months. No effect of the desiccants was observed on the multiplex ICA strips stored in poly_bags at room temperature with respect to the detection of all three pathogens. In contrast, storing the multiplex ICA strips with oxygen absorber only in poly_bags at room temperature reduced detectability against all three pathogens compared to the strips stored in the absence of both desiccants and oxygen absorbers. In the multiplex ICA strips stored with both desiccants and oxygen absorbers, no synergistic effect was observed; however, the reduction in detectability seen during storage with only the oxygen absorbers was prevented. No effect of the vacuum state was observed on strips stored at room temperature for the detection of all three pathogens, except for those stored with oxygen absorbers only at 6 months.

Table 1. Reactivity of multiplex immunochromatographic assay (ICA) strips for MHV, HVJ, and Tyzzer detection in mice in different stored conditions.

		In polyethylene zipper-sealed bags												In vacuum-sealed polyethylene bags
Desiccants		-			+			-			+			-			+			-			+
Oxygen absorbers		-			-			+			+			-			-			+			+
Duration of storage (month)		6	18	36	6	18	36	6	18	36	6	18	36	6	18	36	6	18	36	6	18	36	6	18	36
18–28°C (Room temperature)	HVJ*	+	+	-	+	+	-	-	-	-	+	+	-	+	+	-	+	+	-	+	-	-	+	+	-
	MHV**	+	+	-	+	+	-	-	-	-	+	+	-	+	+	-	+	+	-	+	-	-	+	+	-
	Tyzzer***	+	-	-	+	-	-	-	-	-	+	-	-	+	-	-	+	-	-	-	-	-	+	±	-
4°C	HVJ	+	+	+	+	+	+	±	+	±	+	+	+	+	+	+	+	+	+	+	-	+	+	+	+
	MHV	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
	Tyzzer	+	±	±	+	±	±	-	±	-	±	±	+	-	±	+	-	±	+	+	±	±	+	±	+
−30°C	HVJ	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
	MHV	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
	Tyzzer	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	±	+	+	±	+	+	+	+

HVJ*, Sendai virus, previously also known as hemagglutinating virus of Japan; MHV**, mouse hepatitis virus; Tyzzer***, Tyzzer’s disease, the pathogen of this disease is Clostridium piliforme; +, The intensity of both the control and test lines against each positive serum was clear. It was regarded that detectability of the ICA strip was maintained; ±, The intensity of both the control and test lines in each positive serum were reduced, making evaluation difficult. It was regarded that detectability of the ICA strip was not maintained; -, Neither the control nor test lines against each positive serum was detected, the control line was detected but the test line was not detected, or the test line was detected but the control line was not detected. It was regarded that detectability of the ICA strip was not maintained.

The intensity of immunoreactive bands for both Tyzzer and the control showed a decrease when the multiplex ICA strips were stored at 4°C in poly_bags without both desiccants and oxygen absorbers after 18 months (Table 1). Therefore, it was regarded that the detectability of Tyzzer and the control lines was maintained for up to 6 months after storage. However, the detectability of the HVJ, MHV, and control lines maintained for up to 36 months. When the strips were stored at 4°C in poly_bags, no effect of the desiccant was observed in the detection of MHV, HVJ, and Tyzzer. Conversely, storing the multiplex ICA strips with oxygen absorbers only in poly_bags at 4°C reduced detectability against HVJ and Tyzzer. No synergistic effects were observed when the strips were stored with both desiccants and oxygen absorbers, although the reduction in detectability of HVJ seen during storage with only the oxygen absorbers was prevented. Under vacuum storage conditions, the detectability of HVJ and Tyzzer showed no consistent advantage over poly_bag storage. The intensity of all immunoreactive bands for the control and test lines regarded as that the detectability was maintained at 36 months after storage were the same with the intensity of the control and test lines before storage (Fig. 3).

Fig. 3.

Representative reactivity of multiplex immunochromatographic assay (ICA) strips after 36 months of storage for detection of hepatitis virus (MHV), hemagglutinating virus of Japan (HVJ), and Tyzzer in mice. The following antibody-positive sera for each infectious disease in mice were used: A) Sendai virus, previously known as HVJ, B) MHV, and C)Clostridium piliforme, the pathogen of Tyzzer’s disease (Tyzzer), diagnosed using ELISA and an immunofluorescent antibody assay. Serum (0.75 μL) was diluted with 150 μL of phosphate-buffered saline (PBS). The strips in polyethylene zipper-sealed bags (poly_bags) were stored at room temperature in a desiccator for 36 months. The following storage conditions of the desiccant and the oxygen absorber were set in poly_bags and in vacuum-sealed poly_bags (vacuum_bags): 1) without both desiccants and oxygen absorbers; 2) with desiccants only; 3) with oxygen absorbers only, 4) with both desiccants and oxygen absorbers.

The multiplex ICA strips in poly-bags stored at −30°C showed clear control and test lines with immune sera against all three pathogens even 36 months after storage, regardless of the presence or absence of desiccants or oxygen absorbers (Table 1). The multiplex ICA strips stored in vacuum_bags maintained the detectability, except for a decrease in the immunoreactivity band strength of the Tyzzer and control lines when stored with desiccants or oxygen absorbers after 18 months. The intensity of all immunoreactive bands for the control and test lines regarded as that the detectability was maintained at 36 months after storage were the same with the intensity of the control and test lines before storage (Fig. 3).

No apparent nonspecific reactions were observed in the tested samples.

Evaluation of strip reactivity of the multiplex ICA strips for detection of HVJ, SDAV, and Hanta in rats under each storage condition

As shown in Table 2, the intensity of immunoreactive bands in multiplex ICA strips stored in poly_bags for 18 months without both desiccants and oxygen absorbers at room temperature were reduced for Hanta and the control. Therefore, it was regarded that the detectability of Hanta and the control lines was maintained for up to 6 months after storage. However, the detectability of HVJ, SDAV, and the control was maintained for up to 18 months. No effect of the desiccant was observed on the multiplex ICA strips stored in the poly_bags at room temperature for the detection of all three pathogens. In contrast, multiplex ICA strips stored with oxygen absorbers only in poly_bags at room temperature showed a reduction in detectability against all three pathogens. In multiplex ICA strips stored with both desiccants and oxygen absorbers, the reduction in detectability observed for HVJ and SDAV during storage with only the oxygen absorbers was prevented, whereas the reduction in detection of Hanta remained unaffected. In vacuum storage, the detectability of the multiplex ICA strips for the following pathogens was lower than that when stored in poly_bags: Hanta stored without both desiccants and oxygen absorbers after 6 months, HVJ stored with both desiccants and oxygen absorbers after 6 months, and SDAV stored with desiccants only and with both desiccants and oxygen absorbers after 18 months.

Table 2. Reactivity of multiplex immunochromatographic assay (ICA) strips for detection of HVJ, SDAV, and Hanta in rats in each stored condition.

		In polyethylene zipper-sealed bags												In vacuum-sealed polyethylene bags
Desiccants		-			+			-			+			-			+			-			+
Oxygen absorbers		-			-			+			+			-			-			+			+
Duration of storage (month)		6	18	36	6	18	36	6	18	36	6	18	36	6	18	36	6	18	36	6	18	36	6	18	36
18–28°C (Room temperature)	HVJ*	+	+	-	+	+	-	±	-	-	+	+	-	+	+	-	+	+	-	±	-	-	±	+	-
	SDAV**	+	+	-	+	+	-	±	-	-	+	+	-	+	+	-	+	±	-	±	-	-	+	±	-
	Hanta***	+	±	-	+	-	-	-	-	n.d.	±	±	-	±	±	-	±	±	-	-	-	-	±	±	-
4°C	HVJ	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
	SDAV	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	±	+	+	+	+
	Hanta	+	±	+	+	+	+	±	±	+	±	+	+	+	-	+	+	+	+	±	-	+	±	±	+
−30°C	HVJ	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+
	SDAV	+	+	+	+	+	+	+	+	+	±	+	+	+	+	+	+	+	+	+	+	+	+	+	+
	Hanta	+	+	+	+	+	+	+	+	+	±	+	+	+	+	+	+	+	+	±	+	+	+	+	+

HVJ*, Sendai virus, previously also known as hemagglutinating virus of Japan; SDAV**, Sialodacryoadenitis virus; Hanta***, Seoul type orthohantavirus; n.d., not determined because the strip did not function properly; +, The intensity of both the control and test lines against each positive serum was clear. It was regarded that detectability of the ICA strip was maintained.; ±, The intensity of both the control and test lines in each positive serum were reduced, making evaluation difficult. It was regarded that detectability of the ICA strip was not maintained.; -, Neither the control nor test lines against each positive serum was detected, the control line was detected but the test line was not detected, or the test line was detected but the control line was not detected. It was regarded that detectability of the ICA strip was not maintained.

In multiplex ICA strips stored in poly_bags at 4°C, the detectability of HVJ, SDAV, and the control was maintained for up to 36 months after storage, regardless of the presence or absence of desiccants or oxygen absorbers (Table 2). In contrast, the immunoreactive bands of Hanta were reduced or not detected at 6 and 18 months after storage, except during storage with desiccants only. No effect of the vacuum state was observed on strips stored at 4°C for the detection of all three pathogens. The intensity of all immunoreactive bands for the control and test lines regarded as that the detectability was maintained at 36 months after storage were the same with the intensity of the control and test lines before storage (Fig. 4).

Fig. 4.

Representative reactivity of multiplex immunochromatographic assay (ICA) strips after 36 months of storage for the detection of hemagglutinating virus of Japan (HVJ), Sialodacryoadenitis virus (SDAV), and Hanta in rats. The following antibody-positive sera for each infectious disease in rats were used: A) Sendai virus, previously also known as hemagglutinating virus of Japan (HVJ), B) Seoul type orthohantavirus (Hanta), and C) SDAV diagnosed using ELISA and an immunofluorescent antibody assay. Serum (0.75 μL) was diluted with 150 μL of phosphate-buffered saline (PBS). The strips in polyethylene zipper-sealed bags (poly_bags) stored at room temperature in a desiccator for 36 months. The following storage conditions of the desiccant and the oxygen absorber were set in poly_bags and in vacuum-sealed polyethylene bags (vacuum-bags): 1) without both desiccants and oxygen absorbers; 2) with desiccants only, 3) with oxygen absorbers only, 4) with both desiccants and oxygen absorbers.

The multiplex ICA strips stored in the poly_bags and vacuum_bags at −30°C showed clear control and test lines with immune sera against all three pathogens in most storage conditions, except in the poly_bags with both desiccants and oxygen absorbers and in the vacuum_bags with the oxygen absorbers only at 6 months after storage (Table 2). The intensity of all immunoreactive bands for the control and test lines at 36 months after storage were the same with the intensity of the control and test lines before storage (Fig. 4).

No apparent nonspecific reactions were observed in the tested samples.

DISCUSSION

The storage of ICA strips is vital for implementing rapid response measures during an infectious outbreak, since the preparation of ICA strips is time-consuming and labor-intensive. Therefore, this study aimed to determine the optimal long-term storage conditions for the developed multiplex ICA strips for the simultaneous serodetection of MHV, HVJ, and Tyzzer in mice, and Hanta, HVJ, and SDAV in rats. We demonstrated that the multiplex ICA strips maintained detectable reactivity for all six target pathogens for 36 months when stored in poly_bags at −30°C, regardless of the presence or absence of desiccants, oxygen absorbers, or vacuum conditions.

Although there are numerous reports on ICA using colloidal gold conjugates, there are few reports on the storage conditions and stability of ICA strips. There are reports on the storage conditions and stability of ICA strips using colloidal gold conjugates, although the detection targets are different [3, 4, 7, 9, 14, 17, 20,21,22, 24,25,26,27,28]. It has been reported that the detectability of strips stored at room temperature is maintained for at least 2 months and up to 6 months, and at 4°C, for at least 2 months and up to 12 months. Although there have been reports of ICA strips stored in sealed plastic bags, the ICA strips in these reports were stored with desiccants, and there are no reports comparing storage conditions without desiccants [4, 14, 20, 21, 24]. The zeolite used in this study, which is one of the desiccants, showed no effect on the detectability of HVJ, MHV, Tyzzer in mice at either room temperature or at 4°C storage. In the detection of HVJ, SDAV, Hanta in rats, no effect of the zeolite was observed, except for Hanta at 4°C storage. These results suggest that storage with zeolites is not a critical factor for maintaining the detectability of ICA strips. Furthermore, it is necessary to examine other desiccants, such as silica gel and activated alumina to determine their effectiveness.

In multiplex ICA strips stored in poly_bags with an oxygen absorber at room temperature, detectability for all six targeted pathogens was reduced compared with storage in the absence of both desiccants and oxygen absorbers. The oxygen absorber used in this study prevents oxidation of the stored items by creating an oxygen-free environment inside the storage container by utilizing the oxidation reaction of iron powder. If the cause of the reduction in the detectability of the multiplex ICA strips is an oxygen-free environment, the detectability of the multiplex ICA strips stored in vacuum will also be reduced. However, the multiplex ICA strips stored in vacuum_bags at room temperature without both desiccants and oxygen absorbers had the same detectability as the poly_bags without both desiccants and oxygen absorbers. Therefore, the reason for the reduced detectability of the multiplex ICA strips stored in the poly_bags with oxygen absorbers at room temperature is not the absence of oxygen. In the multiplex ICA strips stored in poly-bags with both desiccants and oxygen absorbers, no reduction in detectability was observed. These results suggest that moisture is necessary for the oxidation reaction, which was absorbed by the desiccants, therefore inhibiting the oxidation reaction. On the other hand, the oxygen absorbers used in this study generate heat when they react with oxygen. The heat generation by the oxidation reaction might have caused denaturation of antibodies and antigens on the strips, resulting in a possible decrease in the detectability. Moreover, condensation might occur around the oxygen absorber by the heat generated during oxidation reactions. Therefore, the reduction in the detectability of ICA strips stored in the poly_bags with oxygen absorbers only at room temperature may be related to factors such as heat and condensation generated during oxidative reactions. Further studies are needed to investigate the cause of the reduction in the detectability of ICA strips stored in poly_bags with oxygen absorbers at room temperature.

Previous reports indicated that the detectability of the ICA strips was maintained longer when stored at 4°C than at room temperature, regardless of the storage container. This corresponds to the findings of this study, as the detectability of the multiplex ICA strips was better maintained at lower storage temperatures, regardless of the presence or absence of desiccants, oxygen absorbers, or vacuum conditions. Although there have been no reports of storing ICA strips at −30°C, this study demonstrated that ICA strips stored in poly_bags at −30°C maintained detectability for all six targeted pathogens for 36 months.

There are some limitations of this study. Firstly, the ICA strips, desiccants, and/or oxygen absorbers should have been stored in properly separated conditions to avoid the effects caused by mutual contact. Another limitation is that it would be preferable to use double-sealed bags to reduce the amount of oxygen in the experimental samples. After solving the above issues, further investigation by using a large number of specimens is needed to determine the sensitivity, specificity, and reproducibility of the ICA strips stored at −30°C in poly_bags.

This study showed that storing ICA strips in poly_bags at −30°C, which is a simple method, is able to maintain detectability long-term for the strips format and pathogen targets examined in this study.

CONFLICT OF INTEREST

The authors declare no conflicts of interest in association with this study.

Funding Statement

This work was supported by a Grant-in-Aid for Science Research from the Japan Society for the Promotion of Science (20K06456).