text: This invention relates to a method for quantitatively, semi-quantitatively, or qualitatively detecting a target substance in a predetermined volume of a liquid sample. This invention also relates to a device for measuring the target substance in the liquid sample used for carrying out said method, which has a structure capable of collecting a predetermined volume of the liquid sample.
  Various methods for measuring a target substance in a liquid sample by allowing a reaction of said target substance on a solid phase are currently available.
  Typical examples of such methods include methods wherein a target substance in a liquid sample is physically adsorbed on a solid phase, a binding substance which specifically binds to the target substance to be measured is bound to the target substance adsorbed on the solid phase, and amount of the binding substance bound to the target substance to be measured is determined by means of radioactivity, color development, fluorescence, luminescence, and the like.
  In other typical methods, a reactive substance which specifically reacts with the target substance in the liquid sample or the binding substance is immobilized on a solid phase, the liquid sample is applied onto the solid phase on which the reactive substance or the binding substance has been immobilized, and the amount of the target substance in the liquid sample is determined by means of the reaction between the target substance and the reactive substance or the binding substance.
  In the methods utilizing the binding substance, binding reactions such as antigen-antibody reaction, nucleic acid hybridization reaction, and receptor-ligand reaction are carried out on the solid phase by using binding substances such as antigen, antibody, antibody fragment, nucleic acid, lectin, receptor, and ligand.
  The methods utilizing the reactive substance include enzymatic methods wherein an enzyme whose substrate is the target substance in the liquid sample is immobilized on the solid phase, the liquid sample is applied to the solid phase on which the enzyme has been immobilized, and concentration of the target substance in the liquid sample is measured by detecting the enzymatic reaction on the solid phase between the enzyme and the substrate which is the target substance. Typical enzymatic measurements include measurements of glucose by using glucose oxidase and peroxidase, urea nitrogen by using urease and indophenol, cholesterol by using cholesterol oxidase and cholesterol esterase, GOT by using malate dehydrogenase and NADH, and GPT by using lactate dehydrogenase and NADH, which are mainly utilized in urinalysis and hemanalysis.
  The methods for measuring the target substance in the liquid samples using the above-mentioned reactions on the solid phase are widely employed in such applications as clinical examinations because of their simple measuring procedure as well as high accuracy, specificity and sensitivity. Among such methods, those utilizing sheet-like solid phase are particularly useful since they are capable of measuring the target substance in a minute amount of liquid sample as well as accurately measuring liquid samples such as nipple discharge whose properties including viscosity and protein concentration are, unlike urine or serum, significantly different from sample to sample.
  A typical method for measuring the concentration of a target substance in a liquid sample by utilizing the reaction on a sheet-like solid phase is disclosed in Japanese Patent Application No. 63-252357 filed by the inventors of the present invention. This method relates to a method for detecting a target substance such as tumor-associated antigen, particularly CEA (carcinoembryonic antigen), in a minute amount of nipple discharge. This method has enabled a detection at an early stage of mammary cancer in patients exhibiting abnormal secretion of nipple discharge. In particular, this method has enabled a detection at a quite high sensitivity of mammary cancer with no tumor, which is likely to be missed by palpation or visual examination in cancer screenings. This method has also enabled a simple and accurate measurement.
  The method disclosed in Japanese Patent Application No. 63-252357, supra has also enabled to measure concentration of the target substance in such sample as nipple discharge, whose viscosity varies from sample to sample and which is collected not more than about 10 .mu.L at a time. In this method, the liquid sample is applied to a relatively large area on the sheet-like solid phase, and therefore, the sample contacts with a larger amount of the substance such as antibody which specifically binds to the target substance to be measured. Thickness of the thus applied sample liquid phase to be measured may also be decreased to increase the reactivity and minimize effects on the measurements of the properties of the sample such as viscosity. Accordingly, this method may be utilized in such cases wherein sample collection using conventional collecting devices including pipette and capillary is difficult to carry out and wherein the sample volume is to small to measure. Non-specific adsorption of the non-specific constituents in the sample onto the solid phase is also suppressed by selecting an appropriate material for the sheet-like solid phase.
  Since the non-specific constituents included in the sample may be easily removed by washing the solid phase either before or after the drying of the liquid sample on the solid phase irrespective of the viscosity of the sample, properties of the sample as well as the drying of the sample least affected the results obtained by this method. This is a marked advantage for such scene as mass screening wherein a large number of samples are occasionally collected, since once collected and dried samples may be left as they are until a sufficient number of samples are collected to resume subsequent procedure.
  In most of the prior art methods for measuring the concentration of the target substance in a liquid sample, a sufficient volume of the liquid sample is collected to either immerse the reaction area of the solid phase therein or apply the sample on the reaction area although the volume of the liquid sample required may differ from method to method, and the reaction is then promoted either on the solid phase immersed in the liquid sample or on the solid phase onto which the liquid sample has been applied to determine the concentration of the target substance by means of the thus promoted reaction.
  When the reaction on the solid phase depends on the concentration of the target substance in the liquid sample and not on the volume of the liquid sample, namely, the absolute quantity of the target substance in the sample, any desired volume of the liquid sample may be brought into contact with the liquid phase so long as the volume of the liquid sample is sufficient for immersing the reaction area of the solid phase in the sample or applying the sample onto the reaction area.
  This is the case for some of the reagents utilizing an enzymatic reaction on the solid phase for their measurements, for example, urinalysis reagents wherein the solid phase is immersed in any desired volume of urine collected in a cup so that the enzymatic reaction may take place on the solid phase.
  On the other hand, when the reaction on the solid phase depends not only on the concentration of the target substance in the liquid sample but also on the amount of the liquid sample, namely, the absolute quantity of the target substance in the sample, a predetermined volume of the sample should be brought into contact with the solid phase. This is the case for some of the reagents utilizing an enzymatic reaction on the solid phase and most of the reagents utilizing a binding reaction on the solid phase for their measurements.
  As set forth above, simple, convenient methods are known for measuring a target substance in a liquid sample.
  These prior art convenient measurements may be employed for the cases wherein the reaction on the solid phase depends solely on the concentration of the target substance in the liquid sample and not on the amount of the liquid sample, namely, the absolute quantity of the target substance in the sample. Even when the reaction depends on the amount of the liquid sample, such prior art measurements are sufficient for limited applications wherein either qualitative results in the form of positive/negative determination or semi-quantitative results are the only results pursued since the amount of the liquid sample collected is not so critical in such measurements.
  When a predetermined volume of the liquid sample must be collected for quantitative measurements of the target substance, an additional step of measuring a predetermined volume of the liquid sample would be necessary for each of the sample collected, requiring devices such as pipette and measuring cup for dispensing the sample. When a large number of the liquid sample are to be measured, the time consumed for such a troublesome step becomes non-negligible to result in a decreased efficiency of the measurements. When the volume of the sample collected is minute, in particular, such an additional step should be carried out accurately with great care. Moreover, when properties of the sample such as viscosity are inconsistent, or when a precise collection of the sample by pipette or capillary is difficult due to high viscosity, the step of collecting a predetermined volume of the liquid sample may become a serious obstacle for the measurement.
  As set forth above, there is a strong demand for the development of a measurement allowing for a substantially constant volume of the liquid sample to be conveniently collected onto the solid phase and allowing for the target substance to be determined at any desired time either before or after the drying or solidification of the collected liquid sample on the solid phase, especially in such a scene as mass screening wherein a large number of the liquid samples are to be collected, and particularly for the collection of such a sample as nipple discharge wherein a minute volume of the sample having inconsistent properties is to be collected.
  To fulfill the above-described social requirements, the inventors of the present invention have carried out an investigation to develop a method and a device capable of collecting a predetermined volume of the liquid sample even when the volume of the liquid sample is minute as well as capable of measuring the volume of the target substance in the liquid sample by a simple operation, and found a structure capable of retaining a substantially constant volume of the collected liquid sample within the reaction area in course of measuring the target substance in the liquid sample by means of a reaction on the solid phase. The present invention was completed on the basis of such a finding.
  It is accordingly an object of the present invention to provide a method for collecting a predetermined volume of a liquid sample to quantitatively, semi-quantitatively, or qualitatively detect a target substance contained in the liquid sample.
  Another object of the present invention is to provide a device for measuring the target substance in the liquid sample for carrying out said method, comprising a structure capable of collecting a predetermined volume of the liquid sample.