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Anigen Rapid Canie Heartworm Ag 2.0 Test Kit
	RG 11-02
	The qualitative detection of Canine Dirofilaria immitis antigen in canine serum, plasma, or whole blood.

- Ten(10) Rapid HeartWorm Ag Test Kits.
- Ten(10) Disposable droppers for specimen.
- One(1) Instruction for use.
- Ten(10) Anticoagulant tube.

[Method]

1. Specimen Collection and Preparation
1) The test should be used to serum, plasma, or whole blood (anticoagulated).
[Whole blood]
(1) Blood should be collected by a disposable syringe to tube treated with anticoagulant (Heparin, EDTA, or Citrate).
(2) Collected blood should be tested immediately or for up to 4 hours at room temperature. It may be stored at 2~8℃ for 24 hours.
(3) Severe hemolytic blood may affect the result.

[Plasma]
(1) Blood should be collected by a disposable syringe to tube treated with anticoagulant (Heparin, EDTA, or Citrate). and then separate plasma by centrifugation.
(2) Plasma may be stored at 2~8℃ for up to 2 weeks, for longer storage (1 year), freeze at of below -20℃.

[Serum]
(1) Blood should be collected by a disposable syringe to tube non-treated anticoagulant (Heparin, EDTA, or Citrate).
(2) Collected blood should be left at room temperature for 30minutes for coagulation. And then separate serum by centrifugation.
(3) Serum may be stored at 2~8℃ for up to 2 weeks, for longer storage (1 year), freeze at or below -20℃.
2) If the test kit and specimens are stored refrigerated, they should be brought to room
temperature prior to use.

2. Procedure of the test
1) Remove the test device from the foil pouch, and place it on a flat and dry surface.
2) Collect some sample using the disposable dropper.
3) Add 2 drops (approximately 80ul) of canine serum, plasma or whole blood into the sample hole.
4) As the test begins to work, you will see purple color move across the result window in the center of the test device.
5) Interpret test results at 5 ~ 10 minutes. Do not interpret after 10 minutes.

[Interpretation]

1) Negative result
The presence of only one band within the result window indicates a negative result.
2) Positive result
The presence of two color bands (“T” and “C”) within the result window, no matter which band appears first indicates a positive result.
3) Invalid Result
If the purple color band is not visible within the result window after performing the test, the result is considered invalid. The directions may not have been followed correctly or the test may have deteriorated. It is recommended that the specimen be re-tested.

1) When can Anigen HW Ag test kit show the false positive result?
The false positive or false negative can be shown at animal clinics.
However, before confirmation of false reaction, the veterinarian should keep in mind the occult infection.

1. In case of early infection stage. Generally, the immunochromatographic assay kit can detect from 8 months because the microfilaria grow up to adults worm for 7 months after infection -> false negative can be occurred.

2. In case of unisex infection (male or female only infection) to the dog. In this case, the microfilaria can not be produced in the blood and can not be observed under the microscope. -> false positive can be occurred.

3. In case of Ivermectin treatment (the Ivermectin can kill the microfilaria), the microfilaria can not be shown in the blood -> false positive can be occurred.

4. By the immune response of the body, the microfilaria can be disappeared -> false positive can be occurred.

5. When the customer read the result after 20 minutes, the result can change from negative to false positive..
So, we wrote the following sentences in the insert sheet.
"5) Interpret test results at 5 ~ 10 minutes. Do not decide after 20 minutes"

Therefore, the final diagnosis should be considered with clinical signs, X-ray, knott method, or other method together.
The specificity of Anigen HW kit is from 98.2% to 100%.

2) What antigen is detected by ANIGEN HW Ag test kit (metabolite of HW or anything)?
Anigen HW Ag kit detects 14 KDa Ag of D. immitis.
The 14 KDa Ag is common antigen of circulating microfilaria and adult female and male worm.
The monoclonal antibody against 14 KDa Ag of D. immitis is using as detector and captures materials of HW Ag kit.

3) Why can ANIGEN HW Ag test kit detect all male, female and microfilaria?
Because 14 KDa Ag is common antigen for circulating microfilaria and adult female and male worm.
But even though our kit use the 14 kDa monoclonal antibody, it is very hard to appear the positive results in microfilaria, L3, L4 and L5 stage serum because of the antigen concentration.

4) If the test checked metabolite antigen, can the test result confuse with canine dipetalonema?
Because ANIGEN HW kit detects 14 KDa Ag of Dirofilaria immitis in blood specimen, it has no cross reaction with canine dipetalonema.

5) No Migration
Especially, CHW Ag test kit is required 2 drops of canine serum, plasma or whole blood. If the dropping volume may be inadequate such as clotted whole blood sample, the migration will not appeared within the testing time(10 minutes)

6) “Agen”(Synbiotics) speaks that they can only detect female adult worm. For that reason, if they use Agen kits which detect only female, if it is positive they will start a with immiticide treatment.

Once the treatment end, they test again, and if Agen test is negative, they will start heart worm prevention.

But by using Anigen HW kits, If it is positive, can we differentiate whether it is due to adult or due to microfilaria? Then this make them can’t go on with treatment.

1. If Anigen HW Ag kit shows the positive results, the imiticide should be treated immediately. Even though our kit use the 14 kDa monoclonal antibody, it is very hard to appear the positive results in L3, L4 and L5 stage serum because of the antigen concentration. We used 14 kDa monoclonal antibody for detecting the male adult worm as well as the female adult worm, not to detect the microfilaria purpose only. The 14 kDa is the common secretive protein from male worm, female worm and larva stage.

IDEXX kit also uses the monoclonal antibody for detecting the male worm.

2. Most of HW Ag kit (Agen, Anigen, Idexx, Heska etc) can detect the 3-5 adult worms. The advantage of Anigen HW Ag kit is not to detect the larva stage, but Anigen HW Ag kit can detect male adult worm as well as the female adult worm (in the market Anigen HW Ag kit and IDEXX HW Ag kit are these type of kits).

Therefore, Anigen HW Ag kit get sensitivity increased, not show positive result in only larva.

3. In L1 and L2 stage, the larva must be appeared together with the adult worm in the blood. So, it is meaningless to detect the larva only.

Also, in L3, L4, and L5 stage (only larval infection stage), the larva do not secrete sufficient protein for HW Ag test kit to detect the larva in the kit.

4. Additional comment about HW treatment.

Since first treatment of adult worm infection, Anigen HW Ag kit commonly show positive for 1~ 6 months (depending dogs, this is the same with other company kits) because secreted protein would be presenting in the blood even though the adult worm disappeared. The average treatment period is for about 6 months.

7) I used your product Anigen Rapid HW Ag 2.0 test kit on a patient positive for microfilaria, having had two (2) series of imiticide injections two (2) days in a row per series. I rechecked the blood a month after the imiticide injection. Whereas the Ag test showed a NEGATIVE RESULT, the direct microscopic examination revealed a lot of microfilariae present in the peripheral blood.
ANY REASON OR EXPLANATION WHY THIS SHOULD BE?

Your treatment was successful. As you mentioned, I think the imiticide only destroyed the adult worm and the microfilaria has not been affected by the imiticide. Under the microscopic finding, microfilaria will be shown if you did not administrate the Ivomec which kills the microfilaria.

The Anigen Heartworm Ag kit can detect the 14kDa protein secreted from adult worm or microfilaria. But Microfilaria only secretes the small amount of 14kDa protein in the blood. Therefore, our kit may not detect the microfilaria secreted protein after imiticide treatment.

Generally, after imiticde treatment, nevertheless microfilaria present in the blood, our kit may give negative results from 1 month to 6 months according to the individual dog. Conclusively, your treatment was optimal and i recommend you Ivomec treatment in the next time to kill the microfilaria.

8) EDTA volume in the EDTA tube and filling volume of whole blood

EDTA volume in the EDTA tube : 20 ul, filling volume of whole blood: Max. 1ml

9) We have had an incidence of a test on an 18 month old male neuter (ex stray) dog that has tested a VERY FAINT POSITIVE with your test. On repeating test, exactly the same result came up. Is this a definite positive result?

We could answer you that the very faint positive result could be interpreted as positive.

To confirm the result, please do the microfilaria microscopic examination. but we guess that you are hard to find the microfiliaria in the blood smear because the faint test line means the Anigen HW Ag 2.0 kit detected low protein concentrate of HW adult worm in the blood.

10) HW Ag 2.0 kit showed slow migration, it takes 20 minutes. What is wrong?

Regarding slow migration of HW Ag 2.0(transparent covered cassette), we think that it might be occurred by a clotted whole blood sample. As you know, the HW Ag 2.0 doesn't need assay diluent and EDTA anticoagulant tubes for whole blood samples provided in the kit..(The EDTA tube is for whole blood sample.)

In case of HW Ag 1.0, even with clotted whole blood, the assay diluent makes migration, but HW 2.0 could not be.

So, the EDTA tube is required.
If your customers are willing to use EDTA tube even though with fresh whole blood, please let them know this point to avoid same complaint.