Colorimetric determination of somatic cell count in milk

Abstract

The invention involves using a simple colorimetric method for a quantitative test to measure white blood cells in milk samples. The invention includes a new reagent system, a new analysis method, and a new apparatus which permits in-line colorimetric analysis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an embodiment of the in-line apparatus of the present invention.

FIG. 1( a) is a schematic enlargement of the signal portion of the embodiment of FIG. 1 which utilizes light transmission.

FIG. 2 is a graphical representation of the data of Example 1 as summarized in Table 1 of that Example.

FIG. 3 is a schematic enlargement of a preferred signal portion for the embodiment of FIG. 1 in which the light transmission signal system is replaced by a light reflectance signal system.

FIG. 4 is a graphical representation of the data obtained from Example 3.

DETAILED DESCRIPTION OF THE INVENTION

Since over 90% of somatic cells are WBC or leukocytes, the proposed method will directly determine the somatic cell count, yielding quantitative results of individual milk at the cow-side. The proposed analytical system will use an inexpensive photometer and liquid reagents for detection, and will produce accurate quantitative SCC measurements in approximately one minute per assay.

All somatic cells or leukocytes have an enzyme called esterase on their cell wall. The role of the polymorphonuclear leucocytes esterase is to convert acetates to phenols. Over the years, urine test-strips have been used to detect the presence infection by indicating the presence of leukocytes in the urine. However, due to the interferences in sample matrixes such as blood and milk, no field test for leukocytes was available until PortaScience published a new technology in 2004. The novel SCC milk test was based on a solid phase test format, and a new dye substrate, 3-(N-tosyl-L-alaninyloxy)-indol (Taloxin) (U.S. Pat. No. 6,709,868), which is very sensitive to esterase, yielding a strong blue color in the presence of esterase. The enzyme catalyses the hydrolysis of dye-substrate, and forms an indigo blue colored dye as the reaction product. Many other colorless chromogenic esters known in the art may be cleaved by the same enzymatic hydrolysis (U.S. Pat. Nos. 4,278,763; 4,637,979; 4,657,855; 4,716,236; 4,806,423).

The concentration of leukocytes and WBC in milk (SCC) is proportional to the enzyme esterase presence, which is proportional to the end color intensity of the indigo dye. This enzymatic reaction has been commercialized successfully for semi-quantitative measurement of leukocytes in urine (U.S. Pat. No. 4,278,763), and recently a quantitative solid phase cow-side test—the PortaSCC milk test—has also been commercialized (U.S. Pat. No. 6,709,868). Potentially this method is an excellent candidate for the development of an in-line SCC test. However, because of the solubility of the dye substrate and the interferences in the milk sample, no liquid reagent using this principle was ever reported for an in-line application. It was surprising, therefore, to find that we have identified a new surfactant and buffer system that accelerates the reaction and reduced interferences, allowing for a rapid detection of SCC (<90 seconds) in liquid phase. We also found that a simple LED/silicon detector optical system was able to measure the resulting color changes quantitatively, allowing for the first time a simple and inexpensive in-line SCC measurement system to be constructed.

The active reagent of the invention consists of a single colorimetric system that contains a dye substrate and buffer (the preferred embodiment) or two part colorimetric system that contains a dye substrate component and a separate buffer component. The preferred dye substrate used in the reagent system is a member of the indoxyl ester family, such as 3-acetyl indoxyl and 3-(N-tosyl-L-alanyloxy)-indole. However, any known substrate that can be hydrolyzed by the esterase on white blood cells to form a colored dye can be use. The buffer works best at a pH of greater than 9.0, but can be functional between pH 7.0-11.0 and at concentrations between 0.01 M to 2 M. A representative and preferred buffer is Tris(hydroxymethyl)aminomethane, commonly referred to as “Tris”. The dye substrate is dissolved in low molecular weight alcohols such as methanol, ethanol, or isopropanol. A surfactant such as the non-ionic surfactant Triton×100 helps to disperse the cell components in the assay mixture, and many other non-ionic, anionic, or cationic surfactants are suitable for this purpose.

The in-line analyzer of the invention consists of a fluid control system, an optical detection system, and related electronics and display, see FIG. 1. Optionally, a temperature control system can be added to the system.

EXAMPLE 1

Liquid Reagent for SCC Determination

The reagent component of the invention consists of the following formulation:

3-(N-tosyl-L-alanyloxy)-indole 10 mg/mL

Tris buffer  1 molar, pH 9.8 at 24° Celsius
Isopropanol  200 mg/mL
Triton X-100 15 mg/mL

Ten fresh milk samples were collected for this study. One hundred microliters of the reagent is mixed with 100 μL of fresh milk sample, and the color changes measured by a Minolta CR-321 colorimeter in Hunter's units in 180 seconds were plotted against the Deleval's Direct cell counter (DCC) method. The data is summarized in Table 1, and the correlation shown in FIG. 2.

TABLE 1
Correlation of the Present In-Line method versus DCC
		Minolta Color
Sample	SCC by DCC	Change
1	7,000	10.8
2	214,000	12.57
3	382,000	14.22
4	530,000	16.3
5	1,417,000	23.3
6	385,000	15.05
7	790,000	16.85
8	2,445,000	29.45
9	593000	18.31
10	295,000	11.03

EXAMPLE 2

In-Line SCC Determination (Transmittance Mode)

The milk sample from a milking line is introduced to the in-line instrument module by a pump and a series of valves, where it is mixed with the reagent. After a fixed incubation period, the mixture is moved to an optical flow cell, where the color intensity is read. The schematic of the in-line instrument is shown in FIG. 1.

• 1. Fluidic controls—The instrument design has one peristaltic pump 1 and six valves 2 through 7 controlling sample and reagents measurements, mixing, and washing steps required in the assay protocol. The peristaltic pump was selected over direct drive pump because of the proven reliability and low cost. The number of valves can be reduced to three, but using six valves simplifies the design of the sequencing for the initial prototype. Similarly, the number of pumps used can be increased to three or more, and other fluidic controls such as positive displacement syringes can be added to increase the accuracy of the fluidic controls. The instrument also should have available a reagent bottle, a buffer bottle, and a waste bottle.
• 2. Optical detection—Instead of using expensive precision pipetting system for measuring the volumes of fluids, a bubble detector 8 was used to measure exact volumes of samples and reagents. The different segments of fluids was separated by columns of air (bubbles), and by measuring the leading or the ending edges of these bubbles, we were able to measure accurate volumes of fluids with a light emitting diode (LED)based detector inexpensively. However, another simple way of measuring the volumes of fluids was simply counting steps of the motor. An optical flow cell 9 with a path length of 3 mm, an emitter board 10, and a sensor 11, for example, a silicon detector, is used to measure the optical intensity of the color of the reaction mixture. A detailed diagram of the optical module is shown in FIG. 1a. A liquid crystal display (LCD) display 12 displays the SCC as a digital read out. Off-the-shelf electronic control boards were used to control the fluid movements and the signal processing.
• 3. Optional Temperature control—A temperature controlled heating element was designed into the back of the flow cell. The purpose was to keep the assay temperature constant at 37° or 40° Celsius. Since the principle of the reagent is enzymatic based, keeping a constant reaction temperature will ensure the accuracy of the test. A side benefit of running the reaction at slightly elevated temperature is the increase in the reaction rate, which in turn will help decrease the assay time.

The in-line protocol using a flow cell is summarized as follows:

• • (1) A 100 μL sample of milk is introduced into a mixing chamber 13.
  • (2) Next a 100 μL aliquot of buffer/surfactant solution is introduced.
  • (3) Followed by 40 μL of dye substrate.
  • (4) The solution is mixed for 60 seconds in the mixing chamber.
  • (5) The solution is moved to the optical flow cell [9] and read. Color intensity is proportional SCC count.
  • (6) A 500 μL aliquot of buffer washes the flow cell into waste.
  • (7) Steps 1 through 6 are repeated with a 60-180 second turn around for each cow.

EXAMPLE 3

In-Line SCC Determination (Reflectance Mode)

The optical detection module of the in-line SCC instrument was modified using the same flow cell and fluidic controls but the optical detector was changed. The optical signal change was measured by a reflectance mode rather by the transmittance mode. As shown in FIG. 3, the emitter 30 and the sensor were placed on the same side of the optical flow cell. The light source was directed to the flow cell surface by a fiber optics 32, and the reflectance measurement was guided back to the sensor using the same optical fiber bundle. The angle of reflectance measurement was 180 degree in this example, but could be optimized by setting the optical fiber at another angle. The light intensity reflected from the surface of the milk and reagent mixture inside the flow cell 31 was measured. Data were collected for 30,60,90,120, and 180 seconds assay times. A standard curve was constructed using the reflectance mode using the 180 seconds assay time. Seventy fresh milk samples were assayed using this method against the reference laboratory FOSS method, and the correlation plot is shown in FIG. 3.

Claims

1. A reagent system for the colorimetric determination of somatic cell count in milk comprising a dye substrate that can be hydralized by the esterase on white blood cells to form a colored dye dissolved in a low molecular weight alcohol and a buffer in concentrations between 0.01 M to 2M and adapted to maintain the system at a pH in the range of 7.0 to 11.0.

2. A reagent system in accordance with claim 1 additionally including a surfactant capable of dispersing the cell components of milk in an assay mixture.

3. A reagent system in accordance with claim 1 in which said reagent system consists of a mixture of said dye substrate and said buffer.

4. A reagent system in accordance with claim 2 in which said reagent system consists of a mixture of said dye substrate and said buffer and in which said surfactant is admixed with said dye substrate and said buffer.

5. A method for the determination of the somatic cell count in milk comprising mixing a milk sample to be analyzed with a reagent system for the colorimetric determination of somatic cell count in milk comprising a dye substrate that can be hydralized by the esterase on white blood cells to form a colored dye dissolved in a low molecular weight alcohol and a buffer in concentrations between 0.01 M to 2M and adapted to maintain the system at a pH in the range of 7.0 to 11.0, and measuring the colorimetric change in the milk sample.

6. A method in accordance with claim 4 wherein, said reagent system additionally contains a surfactant capable of dispersing the cell components of milk in an assay mixture.

7. An apparatus for the in-line colorimetric determination of the somatic cell count in milk comprising in combination a peristaltic pump, a mixing chamber, a supply of dye substrate reagent, a supply of buffer, means for delivering the dye substrate and said buffer to said mixing chamber, a separate means for delivering a milk sample for analysis to said mixing chamber after said substrate and said buffer have been mixed, and means for measuring colorimetric change of the reagent buffer-milk mixture.

8. An apparatus in accordance with claim 7 in which said means for measuring the colorimetric change is a means for reading color change by the transmission of light through the sample.

9. An apparatus in accordance with claim 7 in which said means for measuring the colorimetric change is a means for reading color change by the reflectance of light from the sample.