About Rapid Kit

1Q: Membrane surface of test slip sometimes shows deterioration like scratch on the surface.
A: On the manufacturing process, we check the scratches on the membrane and separate them from the normal product. However, I think we made a mistake while picking up the scratched membrane, but it doesn't affect test result.
2Q: When I opened the pouch, I saw a yellow line on the T line location. Why does this line appear?
A: Sometimes you might see a yellow line on the T line location because of the capture Ag or Ab that is coated on the membrane. Especially in the case of c. Brucella, the Ab used on this product might cause yellow line. However, nonspecific reactions are not related to that yellow line.
3Q: Faint test line of (CPV Ag) kit are shown during migration and then disappeared in 1~2 minutes. How can I interpret the result?
A: In case of severe diarrhea, the mucus originated from internal surface of intestine is often excreted with feces. Mostly, mucus is sticky and because of its’ stickiness the sample may stop on the test line for a while during migration, and then pass by in a minute. Consequently, you can interpret the result as negative. To avoid this problem, after mixing the mucoid diarrhea feces into the assay diluents, wait 1~2 minutes and then add 4 drops on the sample hole of the cassette.
4Q: The migration was divided into two directions after half of running.
A: It is overflow problem. Do not to use excessive volume of feces and take away sticky material from the feces. Please see the good volume of feces on a swab for CPV, CCV Ag kit. And after mixing the feces and assay diluents, place the bottle on the table for 1 minute until large particles settle down. Then take sample from the upper part inside the bottle excluding feces particles and debris.
5Q: If the kit is stored in freezer(2℃ to 8℃), can we use it right away?
A: The temperature of the test kit and room should be same because of the moisture. So, when you are using the kit, incubate the pouch at room temperature for 30 minutes and then open the pouch of the test kit. Don’t freeze the test kit (below 2℃), but cool storage is OK. The sample will last about 1 year at -20C, and 2-3 years at -80C.
6Q: After 15 mins, the (CPV Ag) showed a weak T line, is it positive?
A: We would like to recommend you to interpret the result within 10 minutes. Because positive result after 10 minutes should be regarded as false positive. Usually negative result can be switched to positive result if the virus titer is low (below detection limit) however, positive result can never be changed to negative result.
7Q: Test sample storage after collection.
A: The sample for the test can be stored for about 1 year at -20C, and 2-3 years at -80C. The feces specimen is stable for 12 hours at room temperature and 48 hours at 2~8’C.
8Q: Malfunction problems: No Control line after testing
A: This phenomenon can be occurred when the buffer is dropped very quickly, i.e. the buffer should be dropped appropriately. If or large volume of buffer is loaded suddenly, they overrun the gold chromogen while migration. Consequently, the C and T lines appear as S shape or are not appeared or are faint.
Conclusively, 4-5 drops of assay diluent should be added slowly, drop by drop.
9Q: All tests were positive even with saline solution and with just water
A: The membrane pad inside the device are coated with gold particles and antigen and possible antibody remaining in some cases. So, there could be positive cases without positive samples as we call it non-specific reaction. That is why you should tell your customer to use assay diluents for the testing.
10Q: What is indication of rapid test for use?
A: Even though the rapid test is less sensitive than PCR (sometimes PCR has a false-positive case though), these tests are very convenient for veterinarians for the accurate, fast, and easy diagnosis. These kinds of products exploiting scientific tools of antigen-antibody binding can help to find exact causative pathogen.
11Q. There are 3 false negative cases here. They confirmed it as CPV based on the post mortem lesion. But our kits showed negative result. Please help me with this.
A: Unlike bacterial infection, Virus infection cannot be differentiated by postmortem lesion, because the lesion is not specific. Virus isolation is needed for further option in this case. If there are 3 false negative results, you should consider another cause of diarrhea. Hemorrhagic gastroenteritis and pancytotropic coronavirus infection should be included as differential diagnosis.
12Q: What kind of method does Anigen rapid test use?
A: The principle of Anigen Rapid test kit is the chromatographic immunoassay. Capture antigen or antibody on the membrane can rapidly detect specific antigen or antibody. It can be used to detect various viruses or bacteria. Once the sample is added on the sample pad, the sample flow on the membrane, and then antigen or antibody in the sample react with capture material on the membrane. Control line confirms if the test is working properly. Specific capture materials and flow technique are very important for our test kit.
13Q: If the test was put below 2℃ or over 30 ℃, is it invalid?
A: Yes, too low or high temperature can affect the result.
14Q: There is no migration. We opened them, some pads are elevated and no migration.
A: This is inferior test kit. In this case, please re-test by another Anigen test kit. If this case occurs again, our distributor will exchange it for new one
15Q: When the color bands (Test and control) show up? and What reaction to present color?
A: The band should be within 5 ~ 10 min. If a band has appeared after 10 min., the result shouldn’t be interpreted. Purple color comes from colloidal gold conjugate (gold-Monoclonal distemper antibody). A purple “Test Line” will be visible in the result window if there are enough distemper virus antigen in the specimen.

Canine Parvovirus

1Q: I want to know parvovirus shedding period in feces.
A: CPV is transmitted by oronasal exposure to virus-contaminated vomitus, feces, and environmental fomites. The virus can also be spread by rodents, birds, and insects. The incubation period ranges from 3 to 14 days but is typically 5 to 7 days. Virus shedding in feces begins 3 to 4 days after exposure during the preclinical incubation period and continues intermittently for up to 2 weeks. Dogs with subclinical infection also shed virus in feces. Thus, infected dogs may be contagious for up to 30 days after exposure
2Q: Pet hospital found the weak positive in CPV/CCV Ag, but negative in CPV Ag. And the dog had no clinical sign. How can I explain this?
A: Basically, the CPV Ag kit and CPV/CCV Ag kit has same sensitivity. There is no difference. Therefore, this difference could be caused by sampling site, time difference. Also, the test result should be interpreted together with the clinical sign. It can be interpreted a suspected one and then test the dog after 2 days later or testing CPV Ab test kit is recommended.
3Q: Specimen Collection and Preparation?
A: 1) Sample from canine or feline feces should be used. 2) The specimen should be tested immediately after sample collection. 3) See the image aside for the recommended amount of fecal sample to make the result accurate.
4Q: Can we use vomit to check CPV Ag?
A: The vomiting is very strong acid. This strong acid can give the false positive reaction. Don’t use the vomit
5Q: Is feces specimen collected from puppy’s diarrhea valid or not?
A: Usually, a watery diarrhea flushes out the parvovirus in the intestinal tract (rectum). So it can make false negative. So, we recommend you to wait for 30 minutes before collecting the feces swab or to collect feces of the ground or the floor of dog’s cage
6Q: For sample taking of CPV antigen test, the fecal sample should include swabbing of the rectal wall or not? Or just feces from the floor is ok?
A: It is not necessary to swab the rectal wall. The CPV is propagated in the crypt cells of vili of the small intestine, and is included in the feces. So the virus is mixed in the feces.
7Q: How about vaccine virus detection?
A: There are several reports that vaccine virus can be detected in the feces by PCR method. So the statement is true that vaccine virus shedding in feces can be occurred. But considering that Anigen rapid Ag test kit is not as much sensitive as PCR method, vaccine interference by rapid test is uncommon case. Plus, there are many commercial CPV vaccines and it is likely to show different vaccine virus shedding competence depending products. In conclusion, vaccine virus detection by Anigen rapid test is not likely, and it depends on the each commercial vaccine.
8Q: Can whole blood, serum or plasma be specimen for detection?
A: Yes, but we recommend you to use anal swap or fresh feces because presence of CPV in blood or serum is limited, and test shows positive only in viremic period. In our instruction for use, blood or serum sample is not included.

Canine Distemper

1Q: Will vaccination influence result? If yes, how long after vaccination can we perform the test? If no, is it because of different strain or other methodology?
A: Vaccine interference (Vaccine virus shedding) effect has been reported. Duration of post-vaccine interference is variable but may last as long as 3 weeks. However, it was confirmed by molecular based assay (Restriction fragment length polymorphism analysis) which is more sensitive than immunochromatographic assay. So Anigen Rapid CDV Test Kit does not likely to detect vaccine virus because virus titer is too low to be detected by rapid kit.
2Q: A dog in other clinic, the CDV Ag was positive, the owner didn’t believe it. So they came to us, the test was negative, after treatment it was tested again and showed positive result. The owner thought it’s our responsibility. Why it comes?
A: We think that sampling had some problems. Sometimes, test results of the same dog show different band density on the same day depending on. In this case, each samples might had different virus titer depending on the sampling site.
3Q: Where is the correct site for conjunctiva swab?
A: For CDV Ag specimen, before a veterinarian collects the conjunctiva swab, swab should be wetted by normal saline. And swab side to side (right and left eyes) in conjunctiva tissue is enough for specimzen sampling.
4Q: What factors would affect the test result?
A: Specimen collection, storage, amount, or procedure of the test could affect the result. Especially, the dropping speed and volume are very important. Please drop one by one with 2~5 seconds interval in every dropping. If a customer take too much sample on the swab, the mixed diluents become denser and denser. These dense diluents hinder the migration of fluid inside cassette. Therefore, we recommend the optimal sample collection. The best sample collection amount is slight smearing on the surface of cotton swab.
5Q: Why there’s no C line in CDV Ag tests?
A: We recommend that test kit should be used according to instructions. Check the sample collection, storage, amount, or procedure of test. In our experience, there is no or faint control line in very low case (about 0.1%) especially, due to the high amount of samples. Generally, this phenomenon is non-specific due to the samples. In this case, retest with another testing method.
6Q: I read the FAQ which indicated that making the swab wet by the assay diluents of CDV Ag kit is wrong, because the assay buffer may cause a problem on dogs when collecting the sample from the conjunctiva by the assay buffered-wet swab. Would this induce chemical irritation or other damage?
A: Yes, Some chemicals in the assay diluents will make irritation to eyes. But the dog cannot say he is irritated on his eye. For that reason, the insert sheet is informing that the swab should be wet using the saline solution. We guess that most of pet clinic keep the saline solution usually
7Q: Some customers tell me that test line appeared 10 minutes after the sample loading, and does that mean the amount of antigen is low or disturbed by other agent? And they always continue asking how they can make the interpretation. If we tell the pet owner that the result is negative, how can we explain the faint test line which had appeared?
A: First, we guess that the virus titer was very low, but the dog is suspected case of CDV positive because the test line appeared within 10 minutes. And if the clinical signs show the CDV infection, it would be considered as CDV positive. If there is no CDV clinical signs, dog should be retested after 3~4 days and if the retesting result is negative, the dog is CDV negative or has recovered from the infection.
8Q: If a dog has been treated with hyper immunoglobulin serum while suffering from canine distemper, would it make difference in your CDV test result?
A: We have experience about hype-immune serum therapy at animal clinics. However, detailed experiments did not carried out. In our experience, CDV antigen in conjunctiva was detected at 1 day after hyper immune sera (SN: above 1024) administration (0.2ml/kg). But, the antigen had disappeared at 5-6 days after administration. In this case, we concluded that IgG did not neutralize the CDV in epithelial cells. That is, CDV can be detected from 1 to 6 days after hyper immune therapy due to the preexisted virus in epithelial cells. Anigen CDV Ag kit can help you to follow disease progress. If the virus disappeared from conjunctiva after therapy (hyper-immune sera, antibiotics etc), regarding to the test result of our kit, the dog may have had survived due to your optimal therapy. If our kit gave a negative result in case of CDV Ag presence (by PCR or other confirmation methods), we could consider it as false negative. However, hyper-immune sera therapy dose not directly influence CDV in conjunctiva. Conclusively, hyper-immune sera dose not influence CDV Ag test results. If the dog gave a negative, it means that the CDV antigen was disappeared in the body, especially in the conjunctiva.
9Q: Sample (Conjunctiva, Saliva) storage after collection and before testing
A: Distemper virus (in conjunctiva, saliva) is unstable. For that reason, after collecting the specimen using cotton swab, the specimen should be immediately extracted and tested. If specimen could not be immediately used, it should be refrigerated at 2~8℃, for the storage not less than 48 hours. And if the specimen is freezeed at -20℃ or below, it will be stable for 1 year.

Canine Heartworm

1Q: I used your Anigen Rapid CHW Ag 2.0 test kit on a patient positive for microfilaria, having had two series of immiticide injections two days in a row per series. I rechecked the blood a month after the immiticide injection. Whereas the Ag test showed a NEGATIVE RESULT, the direct microscopic examination revealed a lot of microfilariae present in the peripheral blood.<
A: Immiticide you`ve treated destroys only adult worm and does not affect microfilaria. So under the microscopic finding, microfilaria will be shown. Microfilaricidal therapy should be considered in this case, because the mechanism of action of microfilaricide is different from that of adulticide. The Anigen Heartworm Ag Test Kit can detect the 14kDa protein secreted from adult worm or microfilaria. But Microfilaria only secrets small amount of 14 kDa protein in the blood. Therefore, our kit will show negative result if the specimen has only microfilaria
2Q: What antigen is detected by Anigen Rapid CHW Ag Test Kit (metabolite of HW or anything)?
A: Anigen Rapid CHW Ag Test Kit detects 14 kDa protein secreted from D. immitis. The 14 kDa Ag is common antigen secreted from circulating microfilaria and adult female and male worm. The monoclonal antibody against 14 KDa Ag of D. immitis is used as detector and captures materials of HW Ag kit.
3Q: There is no migration
A: Anigen Rapid CHW Ag Test Kit use 2 drops of canine serum, plasma or whole blood. If the sample is inadequate as being clotted, the migration will not start within testing time (10 minutes)
4Q: If the test checked metabolite antigen, can the test result be confused with canine dipetalonema?
A: Anigen Rapid CHW Ag Test Kit detects 14 KDa of Dirofilaria immitis antigen which is canine heartworm-specific antigen. So it would have no cross reaction with canine dipetalonema.
5Q: How can Anigen Rapid CHW Ag Test Kit detect all male, female and microfilaria?
A: Anigen CHW Ag kit using Heartworm-specific 14 kDa Ag is a common microfilaria antigen and adult worm including male and female. Even though our kit use the anti-14 kDa Ag monoclonal antibody, it is very hard to detect in microfilaria-infected L3, L4 and L5 stage serum because of low antigen concentration.
6Q: When can Anigen CHW Ag test kit show different result with other diagnostic method?
A: 1. In case of early infection stage: Generally, the immunochromatographic assay kit can detect microfilaria from 8 months of age because normally, a microfilaria grow up to become an adult worm until 7 months after infection -> false negative can be seen. 2. In case of infection induced by only male or female Dirofilaria: In this case, the microfilaria cannot be made in the blood and therefore cannot be observed under the microscope. -> They may show negative result with modified knott test or PCR which detects only microfilaria, but Anigen CHW rapid kit will show is as positive. 3. In case of Ivermectin treatment (the Ivermectin can kill the microfilaria): Microfilaria cannot be shown in the blood -> (Knott test: negative, Rapid test: positive) 4. Because of immune response of body, microfilaria can be disappeared -> (Knott test: negative, Rapid test: positive) 5. When the customer read the result after 20 minutes, the result can change from negative to false positive. So, we would like to recommend you to Interpret test results at 5 ~ 10 minutes. Do not decide after 20 minutes.

E. canis

1Q: I have a doubt about E. canis test. The E. canis rapid test detects only IgG antibodies?
A: Bionote E. canis Ab Rapid test can detect all E. canis immunoglobulin antibodies, both IgG and IgM
2Q: How long does it take for antibodies to develop after E.canis infection?
A: Antibodies can be detected between 7 and 28 days after initial infection. Therefore, dogs with acute ehrlichiosis may have false-negative test results if sufficient time not has elapsed for antibody production to occur, and retesting should be performed 2 to 3 weeks later to demonstrate seroconversion.
3Q: A dog came to our hospital with depression, inappetence, fever, and weight loss. E.canis was positive on rapid test, but owner has complained that the dog has been taken tick control medicine by monthly basis. How can we interpret that result?
A: E.canis prevention relies on tick control with products such as fipronil, amitraz collars, or imidacloprid / permethrin combined with careful searching and prompt removal of attached ticks. Unfortunately, acaricide-resistant strains of Rhipicephalus sanguineus have been reported as a result of indiscriminated use of these drugs. So there is chance of tick infestation. Further confirmatory method is needed in this case.
4Q: E. canis Ab test was strong positive. But clinical signs were mild so I wonder if it is non-specific result.
A: Antibody titer and clinical signs are not related to E.canis infection. For example, sometimes subclinical chronic infection cause extremely high antibody titer (>1: 600,000). These antibodies may persist in the face of treatment, suggesting persistence of the organism. These do not correlate with the severity of hyperglobulinemia, disease in general, or duration of illness. So this case should be treated as E.canis infection and further confirmation is needed.

Feline Panleukopenia

1Q. Causes of False Positive for FPV Ag test kit?
A. 1.In terms of utero infection, the affected litter can be clinically healthy probably due to their innate resistance or the acquisition of maternally derived antibodies(MDAs). Still, these kittens can harbor the virus for up to 2 months after birth. (Csiza et al., 1971a) 2.Not all cats infected with FPV develop clinical signs and the severity depends on age, immune status, and concurrent infections (Foley et al., 1999) 3.Cats can test positive for up to 3 weeks after modified live vaccination (Neuerer et al., 2008) 4.The greatest fecal virus shedding corresponds with the peak of clinical disease, but keep in mind that the virus can be shed for up to six weeks after recovery7 and that subclinical animals can also shed virus. (Greene et al., 1998)
2Q: How about virus shedding period of feline panleukopenia virus for rapid kit use?
A: Virus shedding period of feline panleukopenia infection is relatively short. Unlike parvovirus infection in canine which has 1 month of shedding period, feline panleukopenia virus shedding occurs approximately 2~10 days of exposure. Understanding this difference is important for diagnosis of FPV
3Q: Is canine parvovirus infection possible in cat? And is it also possible that Anigen rapid kit can detect it?
A: Canine parvovirus can infect cat. Clinical signs of CPV infection in cat are almost same with FPV infection but severity is different. Generally, CPV infection in cat is mild and chance of survival is higher than FPV infection. Since the two viruses has high genetic homology, Anigen FPV Ag kit can also detect CPV infection in cat, but Practitioner should aware that CPV infection in cat has very limited virus shedding period (2~5 days), Care should be taken for test result interpretation.
4Q: A kitten came to our hospital for neurologic signs (ataxia). History and other situation indicated FPV infection but rapid test shows negative. Can I believe this result?
A: That`s because of the short virus shedding period of feline panleukopenia virus. If the cat showed neurologic signs, which indicates late period of infection, it is likely that the virus shedding is over. So FPV Ag kit does not reveal the virus infection in this case. There are many causes of neurologic signs in kittens, so consider other causes that should be checked. FPV vaccine history should also be checked.
5Q: How about vaccine effect of FPV Ag kit detection?
A: Unlike canine parvovirus vaccine of dog, feline panleukopenia virus vaccine can affect rapid FPV kit, According to article, there are different virus shedding competence for different commercial vaccine, and detect ability was also different for each commercial kit. It seems like vaccine interference period is about 1 week of post vaccination. So care should be done for result interpretation for FPV diagnosis for cat which vaccinated recently.

Feline Leukemia Virus

1Q: Why do the cats should be tested for FeLV before vaccination?
A: There are two reasons for this. 1. Vaccine interference: There had been some reports about FeLV vaccines causing false positive which is called vaccine interference effects on cats right after vaccination. 2. Useless for already infected cats: Before vaccination, cats should be tested for the FeLV infection in advance because FeLV vaccine to the cats that are already infected with FeLV is ineffective.
2Q: We have positive results with FeLV test for a cat, so we sent the sample to the laboratory for IFA test. But it was negative, how can we interpret the results?
A: Discordant test results occur when results of antigen screening tests and confirmatory IFA tests do not agree, making determination of the true FeLV status difficult. The most common scenario that occurs is like described above. Antigen test showing positive and the IFA test showing negative. In general, antigen tests are considered to be slightly more sensitive (fewer false negatives), and the IFA to be slightly more specific (fewer false positives). With either test, however, the reliability (predictive value) of the test is dependent on the prevalence of infection within different populations of cats. Cats with discordant test results are best considered potential sources of infection for other cats until their status is clarified. The true infection status may eventually be resolved by repeating both tests after 60 days and annually thereafter until the test results agree. Since cats with regressive infection are only transiently antigenemic, some cats are expected to revert to negative status on soluble antigen tests without ever having a positive IFA test
3Q: An intact female cat has brought into our hospital and FeLV was diagnosed. The cat has delivered kittens a few days ago, and we get negative result for all of them. Can we say that the kittens are not infected?
A: Kittens may be tested at any time because passively acquired maternal antibody does not interfere with testing for viral antigen. However, kittens infected as a result of maternal transmission may not test positive for weeks to months after birth. Thus, the test results for kittens are not reliable in this case. Generally, cats should have negative test results for both FeLV and FIV prior to being introduced to group housing. So retest is preferable after few months.
4Q: How long does it take to get FeLV positive result after infection?
A: These tests are usually positive by 30 days after exposure; however, development of antigenemia is variable and may take considerably longer period. Retesting after 30 days is recommended for FeLV suspected case with negative result.
5Q: Will the FeLV Ag kit detect FeLV from vaccinations?
A: Since FeLV tests are based on detection of antigen or DNA and not antibodies, vaccination against FeLV does not generally interfere with testing. However, blood collected immediately following vaccination may contain detectable FeLV antigens or DNA circulating from the vaccine itself. Therefore, it is adequate that samples are collected prior to FeLV vaccine administration. The duration of vaccine interference with testing is unknown.
6Q: There is no migration
A: Especially, FeLV Ag test kit requires 2 drops of assay buffer. If the dropping volume is not enough, the migration will not appear within the testing time (at 10 minutes)

FIV

1Q: Is it possible that Anigen FIV Ab test to differentiate field infection and after vaccination?
A: Anigen Rapid FIV Ab Test Kit differentiate the antibodies induced by natural infection and vaccination both.
2Q: Why FIV test does not have an Ag test?
A: Cats infected with FIV have low levels of viral antigen in their blood, which has prevented the use of in-clinic screening assays based on antigen detection as used in diagnosis of feline leukemia virus (FeLV). However, cats with FIV generally produce high levels of antiviral antibodies. Since FIV produces a persistent, lifelong infection, detection of antibodies in peripheral blood of unvaccinated cats is considered sufficient for routine diagnostic screening.
3Q: Why cats should be tested before vaccination?
A: FIV vaccination induce humoral immunity for antibodies to develop, and normal antibody test does not distinguish vaccination and natural infection.(Our Bionote FIV Ab kit can differentiate the natural infection from vaccination because it only detect the Ab produced by natural infection) In adult case, vaccine history or other DIVA method should be check for misdiagnosis. There are many cases of misdiagnosis of infection in uninfected cats which may lead to inappropriate changes in lifestyle or even euthanasia.
4Q: FIV suspected cat came to our hospital, but rapid Ab test was negative. After that, the cat died. How can we explain this to pet owner?
A: Rapid Ab test may show negative results in very initial or terminal phase. It is known that in case of FIV, the antibody begin to develop after 60 days of exposure in most cats, and development of detectable antibodies may be considerably delayed in some cats. In terminal cases, compromised immune system result in lack of antibody development which may show negative results regardless of severe clinical signs. The latter fits in this case. We recommend you to repeat test minimum of 60 days after the last potential exposure.
5Q: Kitten came to our hospital and owner wanted to test for FIV & FeLV before bringing them to their house. The rapid test result was FIV positive, and FeLV negative. But there`s no clinical signs. Should I consider it infected?
A: Positive FIV antibody tests in kittens are unreliable. Antibodies from FIV-infected or FIV-vaccinated queens are passed to kittens in colostrum. These antibodies often persist past the age when many kittens are adopted into new homes and first tested for retroviral infection. Kittens are considered to be at low risk of infection, even when their mothers harbor the virus. Most kittens that test positive for FIV antibodies are not truly infected and will eventually revert to a seronegative status. It is recommended that kittens with positive FIV antibody tests be segregated from other cats and then undergo retesting every 1 to 2 months. Kittens that remain seropositive at 6 months are considered to be infected. Although postponing testing of kittens for FIV until they are over 6 months of age may be tempting, all kittens and cats should be tested before they are brought into new homes. The vast majority of kittens test negative and can be declared free of FIV infection
6Q: Which Antigen does the Anigen FIV Ab test detects?
A: Our FIV Ab test detects IgG antibodies against FIV.