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DIAGNOSIS OF CANINE HEARTWORM INFECTION | Figure 1. ELISA Test Procedure 1. Anti-CHW antibodies (Ab) linked to an enzyme are mixed with whole blood, plasma, or serum, allowing them to bind to heartworm antigens (Ag) in the test sample. 2. The conjugate–sample mixture is then applied to the sample port (S) and allowed to flow across a membrane on which spots of additional anti-CHW Abs have been bound to detect low (L) and high (H) antigen levels. 3. A wash solution is used to remove unbound enzyme conjugates. The wash is followed by a substrate solution that acts with the bound enzyme to produce a color change, indicating a positive test. 4. Positive (C) and negative (N) control dots are also included on the membrane. Presence of a color change in the positive control is required for a valid test, and sample dots must also have stronger color development than negative control. 5. When no positive control dot develops or the negative control has high reactiv- ity, the test is considered invalid and must be repeated. Antigen Detection nâÞmi-linki`ÊimmÕnoÃoÀLinÌÊ>ÃÃ>ÞÊ - ®ÊÌiVhnol- ogy (Figure 1) was used in the first in-clinic CHW >nÌi}inÊÌiÃÌÃÆÊhoÜiÛiÀ]ÊÃiÛiÀ>lÊVÕÀÀinÌÊin-VliniVÊ 7Ê >nÌi}inÊ ÌiÃÌÃÊ >ÀiÊ immÕnoVhÀom>Ìo}À>«hiVÊ /®Ê assays (Figure 2). Even though they are more sensitive than microfi- laria detection, CHW antigen tests: UÊ >ÞÊ}iÛiÊ v>lÃiÊni}>ÌiÛiÊ ÀiÃÕlÌÃÊ inÊ>nim>lÃÊ Ìh>ÌÊ>ÀiÊ infected only with male worms or with very few vim>liÊ ÜoÀmÃÆÊ >nÌi}inÊ ÌiÃÌin}Ê v>ilÃÊ inÊ ÌhiÃÊ V>ÃiÊ because the monoclonal antibodies used in the tests primarily detect female-specific antigens7 UÊ ÀiÊ noÌÊ >lÜ>ÞÃÊ «ÀiViÃiÊÜiÌhÊ Ài}>À`Ê ÌoÊ VoÀÀil>ÌionÊ between the amount of heartworm antigen in the blood and the number of adult female worms. Commercial CHW antigen tests evaluated during 2001 to 2002 were found to reliably detect at least 84% of CHW infections in which 3 or more female adult worms were present, but sensitivity decreased when fewer than 2 adult females were present.8,9 DNA Amplification nÊ>``iÌionÊ ÌoÊ`iÌiVÌionÊovÊmiVÀovil>Ài>Ê>n`ÉoÀÊhi>ÀÌ- worm antigen, other tests may be useful in diagnosing CHW infection, especially in microfilaria-negative dogs or dogs with low worm burdens. Recently, polymerase chain reaction (PCR) technology has been used for the detection and identification of specific microfilaria in peripheral blood samples. UÊ m«liviV>ÌionÊovÊ ÊvÀomÊViÀVÕl>Ìin}ÊmiVÀovi- laria in a multiplex PCR assay gave reliable positive results for samples containing as few as 4 microfi- l>Ài>Ê«iÀÊm Ê>n`Ê`iÃÌin}ÕiÃhi`ÊD immitis infections from D repens infections.10 UÊ noÌhiÀÊ mÕlÌi«liÝÊ * ,Ê >ÃÃ>ÞÊ Ü>ÃÊ ÕÃi`Ê ÌoÊ `iÃ- tinguish between 6 different species of microfi- lariae in dogs and resolve discrepancies in mor- Figure 2. ICT (Lateral Flow Test) Procedure 1. A drop of the sample (whole blood, serum, or plasma) is added to the sample port (S) of the test device. 2. The sample drop reconstitutes the conjugate of gold particles linked to anti- CHW antibodies (Ab) dried on a pad in the test device. A chase buffer is often used to improve flow. 3. The CHW antigen (Ag) in the sample and the reconstituted conjugate flow down the test strip over 2 specific areas. t 5IF GJSTU BSFB JT B MJOF DPOUBJOJOH BOUJ $)8 "C UFTU MJOF 5 XIJDI JT JNNPCJ- lized on the strip. If CHW Ag is present, the anti-CHW Ab will capture it along with bound particle conjugate, causing a visible line to form. t &YDFTT; DPOKVHBUF NJHSBUFT QBTU UIJT MJOF BOE PWFS UIF DPOUSPM $ BSFB XIFSF conjugate-specific antibodies capture the particle conjugate causing a visible line to form. Positive samples must produce obvious lines in both the test and control sec- tions of the strip for the test to be valid. Negative samples produce an obvious line only in the control section. Invalid tests, indicated by a line in the test area but not the control area, or by no lines in either area, must be repeated. July/August 2011 Today’s Veterinary Practice 33