 accommodate the correct sample size.

Eels passing through the sample tube channel can be counted by breaking a light path as they flow through the channel. The electric eye is placed at the entrance of the sample analyzer head. Eels flowing through the tubing can activate the instrument.

To detect animals the laser dye is coupled to a labeling molecule such as an antibody or a specific ligand such as avidin Protein A Protein G various antigens or solid materials such as ion exchange resins latex beads or other inert non fluorescent structures and use this labeled compound as the reagent tag i.e. DYE LABELING MOLECULE OR SOLID MATERIAL REAGENT TAG

The standard detection techniques are then carried out as described in. is a special application in which detection is accomplished without the interference of any immobilizing matrix or reaction vessel wall. This special detection system eliminates any background emission from materials other than the tag and hence it is more sensitive than the matrix dependent method. By lowering the background to near zero the measurements are more accurate and precise since they do not depend on the quality of materials used in the production of disposable tips. The results are expressed in positive values rather than the negative correlations seen in competitive radioimmunoassays RIA s. In a competitive RIA system the amount of radioactive label seen or counted decreases as the target molecule being detected increases. In the Near Infrared MolecularAssay NIRMA of the present invention an increase in level or concentration of the target molecule directly corresponds to an increase of dye and hence an increase in light emission.

Among all spectrofluorescent methods laser fluorimetry is the most sensitive 1 6. Sauda et al. 1986 demonstrated detection of 10M laser dye with a 3 nW laser. Also there are over 2000 laser dyes commercially available 7 from which one can choose to develop a practical detection system. Therefore a diode laser a photo diode and a laser dye must match in order to make the detection system workable.

Since most biological and other materials do not fluoresce between 700 and 1300 nm 4 it was important to find a useful laser dye in that range which could bind to a specific carrier or labeling molecules and act as a tag without losing its fluorescent properties.

 5 must bind electrostatically to specific proteins such as albumin lipoproteins and gamma globulins.

While polymethine dyes have an absorption range between 600 and 900 nm 4 most are positively charged and thus are not very useful for directly labeling proteins unless the proteins are negatively charged. Some laser dyes such as IR 125 and IR 144 are negatively charged and hence ideal for labeling positively charged proteins 8. A new dye NN382 can also be used since it has good labeling characteristics.

NN382 has been found to be particularly useful for use in molecular marking of animals. This dye has the following formula 

Because wildlife that is to be detected using the system of the present invention is by definition outdoors the system including dyes used therein must be operable under a variety of conditions and at least from about 5 C. to about 20 C. For purposes of the present invention cold refers to temperatures below about 5 C. Of course the temperatures at which assay are conducted depends on the type of sample used. Water based samples are generally assayed at temperatures above about 0 C. so that they are in the liquid state.

The present invention can use two fundamental disposable tip configurations which are defined by two different types of analysis target area ATA. The first type type 1 has an enclosed ATA composed of a solid phase. This type is shown in. The second type type 2 has an ATA which is solid phase free and not enclosed by materials as seen in.

In tip type 1 the material used for the wall of the disposable tip has a low near infrared excitation emission profile in the wavelength of interest. One example of a useful material is Teflon FEP although other materials can be used. Of suitable materials Teflon FEP is preferred. Many other materials have a higher near infrared background or other less appealing characteristics such as carrying larger electrostatic charges on the surface and hence attracting and binding biomolecules of interest or reagent tags which renders the assay less sensitive. The electrostatic charges of the tip must be specially blocked without interfering with the binding characteristics of the plug or matrix. Also since the tips can be used in a sterile environment they must be sterilizable. This is preferably effected by radiation and must not change the properties of any of the materials used in constructing these tips so that the assay is not affected in any way.

The analysis target area ATA can be made of micro ground glass micro glass or plastic beads as well as porous nylon matrix porous composite matrix e.g. nylon and ion exchange resins or a fine mesh or screen surface. These materials have the same near infrared low non specific binding and sterilizable properties as the FEP housing above. Also they have an added characteristic in that they can bind covalently or by other means target or capture molecules used in the system of the present invention. The target molecules are the chemical structures which are bound to the matrix for the purpose of having specific molecules recognize and bind to them when they are in very close proximity. For example the target molecule could be an antigen an antibody a ligand such as avidin concanavalin A protein A protein G etc. or a hapten such as biotin an enzyme substrate or its product a chelate etc. The matrix may not carry any target or captures molecules at first but these may be supplied in an activated form so that the user can attach any ligand of choice to the matrix.

In the reservoir is larger than the uptake channel permitting a bigger sample volume to be collected and tested. In this embodiment internal pressure fittings are used rather than an external one with an O ring as shown in.

Another embodiment of this design is shown in in order to flatten out the bubble in which detection occurs. In this embodiment a post is set close to the tube end. In this case the bubble is hung between the tube end and the post hence increasing the stability of the sample and making surface readings possible with less scatter of the emission wavelength from the reagent tag. A space exists on each side of the post between the post and the internal wall of the tip so that air has access to the uptake channel. When a negative pressure is created the fluid sample can rise through the uptake channel into the reagent trap and past it to the ATA.

These type 2 disposable tips have a trap which is a zone where two competing molecules bind to a matrix. The trap is situated before the end of the specially beveled tube as shown in.

In another embodiment of the present invention the reagent tag is an avidin tag binding to a biotinylated antibody which is attached to a specific antibody to the antigen Ag2 coupled to a solid matrix.

In this embodiment an antigen determination is conducted. The mechanical sequence of events is identical to those for detecting an antibody. The molecular interactions between antibodies are bound to the matrix rather than to the antigen. This can be effected by direct binding of the antibody to the matrix or through linkers and spacers. One type of spacer molecule is avidin. Coupling of avidin to the matrix followed by binding of a specific biotinylated antibody Xa as the capturing molecule represent a universal coupling technique useful in antigen detection. This antibody Xa recognizes and captures antigen X2 which is then recognized and bound by a second antibody reagent tag Xb. Diagram 3 illustrates multiple variations of this theme.

In another embodiment the antigen Ag1 is captured by the matrix bound AG1 specific antibody. The antigen is then recognized and bound by a second biotinylated antibody which is then bound by the avidin reagent tag.

Antibody or antigen determinations can also be made by reagent tags composed of near infrared probes in or on latex particles ion exchange resins or other particulate matter with the appropriate biophysical and chemical characteristics as shown in Diagram 5.

Assays described above are based upon type 1 disposable tips in which the reagent tag is on a matrix surrounded by the tip wall. Another type of assay is based on type 2 disposable tips which increases the sensitivity and decreases error in the assay.

With a type 2 tip a ligand is coupled to the matrix which ligand specifically binds the molecule of interest in a test sample as well as the reagent tag. The reagent tag is the same as the molecule of interest but is labeled with a laser dye such as NN 382. This is a competitive assay in which an antibody b coupled to the matrix specifically recognizes and binds the test sample antigen b1 flowing through the matrix as well as the reagent tag antigen b1 NN 382. In order to measure the amount of antigen b1 in a test sample a calibrated quantity of reagent tag is mixed with the test sample. The amount of reagent tag added to the test sample is just enough to saturate all of the matrix bound antibody b binding sites when no other competitive antigen to the reagent tag is present in the sample being tested. Hence no excess reagent tag flows through the matrix as it is all captured by the antibody on the matrix and appears in the ATA giving a zero reading. The only means by which the reagent tag appears in the ATA is when the reagent tag and antigen b1 from the test sample are mixed. Test sample antigen b1 competes against the reagent tag for the same binding site of antibody b located on the matrix and therefore some of the reagent tag is not bound to the matrix. The free reagent tag flows into the ATA where it is measured.

As increasing amounts of test sample antigen are present in the mixture more and more of the reagent tag appears in the ATA. This method shows that with a very low concentration of test sample antigen b1 one obtains low readings while with equal amounts of reagent and test antigen b1 half the reagent will appear in the ATA bubble. Finally when high concentrations of antigen b1 are tested most of the reagent tag will be found in the ATA