EP1044371A1

EP1044371A1 - Method and apparatus for on-line testing samples

Info

Publication number: EP1044371A1
Application number: EP98962713A
Authority: EP
Inventors: Robert Hans Meloen; Joannes Pieter Maria Langeveld; Gerard Ligtvoet; Wouter Cornelis Puijk
Original assignee: Pepscan Systems BV
Current assignee: Pepscan Systems BV
Priority date: 1997-12-29
Filing date: 1998-12-29
Publication date: 2000-10-18
Also published as: NL1007925C2; WO1999034211A1; AU1788499A; JP2002522746A

Abstract

Method for serologically testing samples such as blood samples and the like, wherein at least one and preferably a series of sub-samples are separated, wherein the at least one sub-sample is subjected to at least one serological test, wherein the at least one test is performed on-line.

Description

Title: Method and apparatus for on-line testing samples.

The invention relates to a method for serologically testing samples such a blood samples and the like.

In, for instance, meat-processing businesses, it is of great importance to be able to establish whether the animals whose meat is to be processed meet prevailing health standards, at least at the time of the rearing period, the lactation period and/or the slaughter. To that end, for instance blood or another bodily fluid of the animals is serologically tested, to establish, for instance, bacteriological or virological infections thereof. It is known to perform such tests by taking blood from the living animals, which blood is accurately examined in a laboratory. The blood sample is separated into a number of different parts, each of the separate parts being subjected to a specific serological test. The results found are combined into one, on the basis of which the health of the animal in question is determined. These data are fed back to the farm where the relevant animal is located, whereby the animal, its flockmates and/or stablemates are or are not released for slaughter, at least for subsequent human or animal consumption.

Further, it is known to take a random blood sample from a slaughtered animal in a meat-processing business, which sample is serologically tested in the above-described manner in a laboratory. On the basis of these data, a whole batch of meat which may come from a plurality of slaughtered animals, is or is not released for slaughter, at least for subsequent human or animal consumption.

In these known methods, a sample is in each case taken to a laboratory, where the different serological tests are performed by different persons, at least in different stages and by different means. This is costly, time-consuming and little effective. There is also the danger of losing the direct connection between the animal from which the sample was taken and the test results. Moreover, the animal in question will often already be further processed before the relevant results are fully available. Furthermore, testing samples of living animals entails the danger of these samples being no longer entirely accurate at the time of the slaughter. In hospitals, blood banks and the like, (blood) samples are tested in a similar manner.

Hence, the object of the invention is to provide a method of the type described in the introduction, wherein the drawbacks mentioned of the known methods are avoided, while the advantages thereof are maintained. To that end, a method according to the invention is characterized by the features of claim 1.

Performing serological tests on-line offers the advantage that the desired results are available relatively fast, while a good connection between the animal whose sample is taken and the relevant sample that is being processed, is maintained. Moreover, a large number of tests can be performed in a fast and simple manner, while the tests for different animals, or at least parts thereof, can be simultaneously performed in series as well as in parallel. Accordingly, on the basis of the data found, further processing of tested meat or any treatment of a tested animal can be controlled. Owing to the parallel, fast, accurate and relatively cheap performance of a series of serological tests and the accurate control of the further processing or treatment on the basis of the measured values found, the health of humans or animals for whom the meat is intended for consumption and/or of the tested animals themselves is protected more effectively than it used to be, all the more because this readily enables serologically testing a large number of animals, if not all animals that are slaughtered, to be slaughtered or treated.

The term 'on-line' should at least be understood to mean testing a sample, in particular a blood sample, taken from an animal or human on the site. In this respect, 'on the site' should at least be understood to include a space suitable for performing serological tests situated at some distance from the location where the sample is taken, to which space the blood samples taken can be conveyed quickly. Thus, for conveyance, pneumatic dispatch can for instance be used, whereby samples may become available for serological tests within minutes.

In an advantageous embodiment , a method according to the invention is characterized by the features of claim 2. In a method according to the invention, a relatively large number of serological tests are performed on-line on sub-samples separated from a sample taken. To that end, the separated sub-samples are pretreated and introduced into a matrix of receiving cavities, while in different receiving cavities, different serological reactions are generated. To that end, the different sub-samples are preferably mixed with different first treatment mediums, after which the formed mixtures of sub-samples are treated with different or the same further treatment mediums. Hence, in different receiving cavities of one matrix, parallel and/or serially different mixtures of sub-samples, first and any further treatment mediums are obtained, resulting in different reactions or couplings of active components. Next, the sub-samples in the matrix of receiving cavities can be further processed and analyzed, as a result of which a complete picture of the measured values found can be obtained for the animal in question, or at least a portion thereof. Each of the receiving cavities filled after the serological test has ended, gives the result of the serological test performed therein, which for each receiving cavity in question in a row and/or column of the matrix may be a different test . This enables a simple comparison of this matrix with other, known matrices, so that the results found for a large number of different tests can be analyzed at once, for instance through pattern recognition. In this context, 'a matrix of receiving cavities' should at least be understood to mean a number of columns and rows of receiving cavities, wherein for two or more rows and/or columns, the number of receiving cavities in each row and/or column may differ from the number of receiving cavities in an adjacent row or column. The position of each receiving cavity within the matrix can be established by means of a coordinate system (for instance row and column number of a receiving cavity) , for the further processing of the measured values. Whenever this is possible and relevant, a column of receiving cavities is in this specification each time referred to as a series of receiving cavities in which a part of the same sub-sample is provided. Hence, when a plurality of sub-samples are processed simultaneously, the separate sub-samples can be inserted in different columns of receiving cavities. However, it will be appreciated that a random distribution of each sub-sample, i.e. not distributed in rows or columns, can also be employed. Accordingly, by means of a chosen coordinate system, it can for each sub- sample be established in which receiving cavity (-ies) it is inserted.

In a particularly advantageous embodiment, a method according to the invention is characterized by the features of claim 4. By using a mixture of at least two conjugates as at least one of the treatment mediums, the particular advantage is achieved that in different receiving cavities in a column of the matrix of receiving cavities, different serological tests can be performed while they are filled with the same sub-sample. This enables the receiving cavities to be filled in a particularly simple manner, while, moreover, it is guaranteed in a simple manner that all tests in a selected column of receiving cavities are carried out with an identically treated sub-sample, which allows an even better comparison. In each receiving cavity, for instance a different further treatment medium can be added. Also, each receiving cavity in a row of the matrix can, for instance, be treated with the same further treatment medium, while the mutual rows are treated with different treatment mediums . In each receiving cavity, there is obtained a specific combination of bound or unbound antibodies, antigens and the like, which may or may not be labeled with labels detectable by an analyzing method employed.

In this context, the term 'conjugate' should at least be understood to mean an element whereby, in a serological test, antigen, such as for instance viruses or species of foreign substances, or the antibodies specifically generated thereagainst , can be demonstrated. In particular, this should be understood to mean labeled immunoreactants . Labels may at least consist of one or more of the following elements :

1. an enzyme, such as, for instance, horseradish peroxidase (HRPO) , alkaline phosphatase, urease, glucose oxidase, dehydrogenase , β-D-galactosidase ;

2. a fluorochrome ; 3. a radioactive element such as, for instance, H³, C¹⁴,

I¹³¹;

4. biotin;

5. gold spheres; 6. carbon spheres; or 7. europium.

Variations hereto and alternatives will be directly clear to the skilled artisan.

In a further advantageous embodiment, a method according to the invention is further characterized by the features of claim 6.

Coating a matrix of receiving cavities with preferably different further treatment mediums such as specific antibodies or antigens, offers the advantage that this can be performed under well-controlled conditions, prior to the performance of a series of serological tests, since the receiving cavities can be pretreated. For a specific series of serological or like tests, a multiplicity of matrices of receiving cavities, to be referred to as matrix plates, can be prepared in a suitable, unequivocal manner, so that during testing, a matrix plate is always present. Thus, arrays of series of serological tests can be performed at high speed and with great accuracy and reproducibility, at relatively low costs. Particularly when such matrix plate is employed in an on-line method according to the invention, this yields the advantage that in a (semi) continuous process, the desired number of serological tests can be performed on meat, blood or other types of samples that are supplied and discharged (semi) continuously.

In this context, the term 'coating' should at least be understood to mean an element to be used in a serological test, whereby antigens, such as for instance viruses or species of foreign substances, or the antibodies specifically generated thereagainst , can be demonstrated, in particular an immunoreactant . Such a coating may for instance consist of : 1. an intact virus particle, or a specific portion thereof; 2. an intact antibody, or a specific portion thereof;

3. a peptide;

4. a protein other than stated under 1-3; or

5. residues, or combinations thereof. Other coatings applicable in the invention will be directly clear to the skilled artisan.

In further elaboration, a method according to the invention is characterized by the features of claim 7.

In this method, through a smearing movement of the pickup part across one or more receiving cavities, a portion of the sub-sample is inserted into the relevant receiving cavity (-ies) . Thus, relatively small receiving cavities can be filled, which would otherwise be difficult, if not impossible, to fill, due to surface tension of the sub- sample. Moreover, due to the smearing movement of the pickup part, an equal amount of sub-sample is each time inserted into the relevant receiving cavity. Indeed, any excess is smeared off over the top edge of the receiving cavity by the pickup part. Thus, filling of the receiving cavities with in each case an accurately equal amount becomes possible in a particularly simple manner, also in the case of small receiving cavities. In this manner, individual receiving cavities can be filled, but also a series of receiving cavities in a column of, for instance, a matrix of receiving cavities as used in a method according to the invention. Due to, inter alia, the fact that relatively small receiving cavities can be filled without any problem, without loss of sub-sample, and that all sub- samples from the same sample are available and can be used for one or more tests, in a method according to the invention, particularly small amounts of sample may suffice, on which a wide variety of tests can be performed. This is particularly advantageous when only a small amount of the sample or the treatment mediums necessary for the tests is available, or when the sample or the treatment mediums are costly or dangerous.

A smearing operation for inserting the sub-sample into the different receiving cavities may of course be performed in different manners and by different means, while the sub- sample may be received in as well as on the relevant pickup part, prior to smearing. Direct contact between the pickup part and the receiving cavities is not necessary, because transfer will already take place through adhesion. The advantage achieved by avoiding direct contact is that no contamination of the pickup part by the receiving cavities, at least the part thereof in which they are received, or vice versa, will occur, so that the pickup part can be employed repeatedly. As a matter of fact, it will be clear that different pickup parts may be used for different columns of receiving cavities or combinations thereof.

In a preferred embodiment, a method according to the invention is characterized by the features of claim 8.

By simultaneously subjecting the different receiving cavities to a suitable analyzing method, a complete picture of the serological test results, and hence of, for instance, viral or bacteriological deviations in the sample, can be obtained very fast. This is particularly the case when a non- invasive method, for instance radiation measurement, is used, since this does not involve contamination of the analyzing device (s), and hence, a plurality of matrices of receiving cavities can rapidly be analyzed one after the other.

In further elaboration, a method according to the invention is moreover characterized by the features of claim 9, in particular of claims 9 and 10. The use of radiation measurement enables a fast and non-invasive measurement. For this, in particular light measurement is suitable, the more so because images thereof can readily be recorded and analyzed, for instance be compared with comparable images of known samples. By recording the known, relevant measured values, for instance of colors, gray values, X-radiation or like measured values, or combinations thereof, of such known samples as total images in a databank, any deviation from the expected pattern can be established for each receiving cavity through a simple image comparison of an unknown sample with these known images, which gives an indication for the result of a specific serological test. As a matter of fact, it will be understood that parallel to the serological tests, other tests can be performed in the receiving cavities as well, for instance control tests, reference tests and the like, such as measurement of the acidity of the sample or other non- serological test values which may influence the serological test values or at least the interpretation thereof .

Supplementing the databank with reference images taken from samples which are known as far as the relevant values are concerned, offers the advantage that thus, a self- learning system is obtained which always connects optimally to the samples to be tested serologically. The use of an algorithm in a calculating device suitable therefor offers the advantage that this is possible in a particularly simple and automatic manner. In a further preferred embodiment, a method according to the invention is characterized by the features of claim 11.

By serologically testing a number of separate samples simultaneously, a multiplicity of samples can be tested for a large number of aspects in a relatively short time, while samples can be tested parallel and/or serially. The advantage thus achieved is that tests can rapidly be performed, preferably on-line, for a large number of variables. The results can be fed directly to a processing apparatus for the meat, to control the further processing.

The invention further relates to an apparatus for serologically testing, on-line, samples such as blood samples and the like, characterized by the features of claim 12.

Such an apparatus makes it possible in a simple manner to perform on-line serological tests in a fast, effective and accurate manner, on a multiplicity of samples and in a relatively short time. Further, the apparatus offers the possibility of treating each of a number of sub-samples with a first treatment medium, and to subsequently distribute them over a number of receiving cavities, while in each receiving cavity, a second treatment medium can be added to the pretreated sub-sample. As a result, it can per receiving cavity be determined which combination of sub-sample, first pretreatment medium and second pretreatment medium is included. This means that in a simple manner, the possibility is provided of performing in each receiving cavity a different serological test, if desired, starting from the same sample. Thus, simultaneously, an picture of a multiplicity of different sample values is obtained. Moreover, an apparatus according to the invention enables a proper and accurate dosing of sample, sub-sample, first and second treatment medium, partly as a result of which the means for analyzing the contents of filled receiving cavities can selectively represent an absolute or relative measured value which is comparable with previously performed serological tests. The use of an apparatus according to the invention therefore offers a high reproducibility .

In a first advantageous embodiment, an apparatus according to the invention is characterized by the features of claim 14, in particular by the features of claims 14 and 15.

By providing the second (and any further) treatment medium in each of the receiving cavities, preferably prior to the insertion therein of the relevant part of a sub-sample, the advantage is achieved that the receiving cavities can be prepared prior to a test cycle, optionally remotely from the apparatus. In particular when the receiving cavities are accommodated in a changeable unit, this enables a fast and effective processing. By providing the second treatment medium in the receiving cavities in the form of a coating, the advantage is achieved that the second (and any further) treatment medium is fixedly connected to the receiving cavities and cannot become detached therefrom unintentionally. Moreover, this yields a proper bond between the coating and elements from the sample which are to be retained from the treated, relevant part of the sub-sample in the receiving cavities, for instance during subsequent rinsing thereof. In each of the receiving cavities, a specific second treatment medium can be included, for instance an antigen or an antibody specific for the serological test which is to be performed at least partially in the relevant receiving cavity.

In a further embodiment, an apparatus according to the invention is characterized by the features of claim 16. The use of a matrix plate in which the receiving cavities are provided offers the advantage of simple exchangeability and processability, while the receiving cavities always have a fixed position relative to each other. By using second pickup means which, through a smearing movement, can introduce a pretreated sub-sample into different receiving cavities, the advantage is achieved that also relatively small receiving cavities can readily be filled with an exact amount of the sub-sample, while in one flowing movement, for instance a column of receiving cavities can be filled. In this context, a 'smearing movement' should be understood to include a relative movement of the pickup part over or along at least one receiving cavity, at such a distance that at least a part of the sub-sample present on or against the relevant second pickup part flows into a receiving cavity at least through adhesion or cohesion.

During the smearing movement, the distance between the second pickup means and the receiving cavities is preferably slightly greater than 0 mm, so that direct contact is avoided and contamination is prevented. The invention further relates to a matrix plate, in particular suitable for use in a method or apparatus according to the present invention, of claim 25. In this context, 'matrix plate' should be understood to mean a carrier provided with a number of series and columns of receiving positions, in particular receiving cavities for receiving during use, in or on each receiving position, a preferably fixed amount of mixture of a part of a sample to be tested and one or more treatment mediums, while of each receiving position the position on the carrier is determinable and fixed unequivocally. Moreover, the advantage is achieved that positioning and dosing can take place in a fast and simple manner. By providing at least a number of the receiving positions, in particular the receiving cavities, with a treatment medium for a mixture to be included in the relevant cavity, the advantage is achieved that dosing thereof is readily possible, while the matrix plates can be prepared for the relevant serological tests prior to use, so that a relatively large number of samples can be processed relatively fast.

In an advantageous embodiment, a matrix plate according to the invention is characterized by the features of claim 26.

Providing a treatment medium in or on a number of the receiving positions on the matrix plate offers the advantage that the relevant treatment medium is fixedly connected to the matrix plate as coating, which simplifies the use and processing of the matrix plates, while, moreover, the retention of components from the mixtures arranged in or on the receiving positions, possibly together with at least a part of the coating, during further treatment with, for instance, buffers, rinsing mediums and the like, can suitably be provided for. By providing different receiving cavities with different treatment mediums, in a matrix plate according to the invention, the advantage can be achieved that on one matrix plate, a multiplicity of tests, in particular serological tests, can be performed simultaneously, while, moreover, the test results of the different tests can be quickly available. The invention moreover relates to an analyzing apparatus according to claim 28.

Such analyzing apparatus has the advantage that a picture of the test results of a series of receiving positions, i.e. of a series of tests performed, can be obtained simultaneously, which results can simply be compared with the test results of a known sample. Preferably, the results of all tests performed can be analyzed simultaneously and compared with the results of known samples . In this respect, non- invasive analyzing method have the advantage of involving no contamination by the samples, so that interim cleaning can be omitted.

The invention further relates to a method for processing meat products according to claim 30.

The on-line testing of samples taken from the animals slaughtered or to be slaughtered, offers the advantage that the test results are available relatively quickly, for instance within sixty minutes, preferably within forty- five minutes, and more in particular within thirty minutes, and in a preferred embodiment within a couple of minutes after the sample has been taken, while moreover, a good, unequivocal coupling between the sample, the animal and the test results can be maintained. Thus, for each animal, an optimal slaughtering and further processing path can be established.

The invention moreover relates to a treatment medium according to claim 32.

The use of a treatment medium comprising at least two specific conjugates has the advantage that a sample treated with this treatment medium can be used for at least two specific tests, in particular serological tests, each conjugate being test-specific.

To clarify the invention, exemplary embodiments of methods and apparatus according to the present invention will hereinafter be further described, with reference to the accompanying drawings .

Fig. 1 schematically shows, in top plan view, an apparatus according to the invention; Fig. 2 schematically shows, in side elevation, an apparatus according to Fig. 1;

Fig. 3 shows, in sectional front view taken on the line III-III in Fig. 1, a matrix plate;

Figs. 3A and 3B show, in sectional side elevation taken on the line III-III in Fig. 1, two alternative embodiments of a matrix plate according to the invention; Fig. 4 shows in an enlarged, sectional side elevation taken on the line III-III in Fig. 1, a receiving cavity of a matrix plate;

Fig. 5A shows, in top plan view according to Fig. 2, the position of the sample-transfer means;

Fig. 5B shows, in top plan view according to Fig. 2, the position of the receiving means for the sample mixed with conjugates;

Fig. 6A shows, in top plan view, application means for inserting the sample/conjugates mixture into the receiving cavity of a matrix plate;

Fig. 6B shows, in sectional side elevation taken on the line VIB-VIB in Fig. 6A, said means during insertion of the mixture into the receiving cavity; Figs. 7A-I schematically show a device for dosing in particular substrates; and

Figs. 8A-D schematically show a device for picking up and dosing liquid.

In this description of the drawings, corresponding parts have corresponding reference numerals.

Fig. 1 schematically shows, in top plan view, an apparatus for serologically testing, on-line, samples such a blood samples and the like according to the invention. This apparatus 1 comprises supply means 2 for the supply of containers 3 which each contain a sample 4, pre-holding means 5, first holding means 6 and second holding means 7, and analyzing means 8, which will all be further discussed hereinbelow.

Each container 3 contains a sample 4 , for instance blood, drawn from a human or animal, which sample 4 is individually recognizable and traceable to the relevant human or animal. The pre-holding means 5 comprise a substantially cube-shaped block 9 which is suspended in bearings 12 for rotation about an axis 11 extending through the centers of two end faces 10 located on opposite sides (Fig. 2) . The other four sides 13 are each provided with a receiving cavity 14 which is slightly cup-shaped and has, for instance, a circular circumference. By drive means 15, for instance a stepping motor and suitable coupling means, the block 9 can be rotated about the axis 11, such that in each case a pre- receiving cavity extends in a horizontal, overlying plane 16. To that end, the block can each time be rotated over 90° or a multiple thereof. Moreover, the drive means 15 are arranged so that the block can be moved back and forth over a relatively small angle of some degrees from the central position shown, for reasons to be further described hereinbelow.

The first holding means likewise comprise a cube- shaped block 17, suspended in bearings 20 for rotation about a second rotation axis 19 extending through two opposite end faces 18 (Fig. 2) . The other four side faces 21 are each provided with five slotted first receiving cavities 22, for instance slightly drop-shaped in top plan view, having a principal direction lying approximately parallel to the axis 19 and each having a first end 23 that is located on, at least adjacent, a central axis 24 of the relevant face, which central axis extends at right angles to the axis 19. Successively the first, third and fifth first receiving cavities 22 have a first end 25A that is located adjacent a first end face 18A, the second and fourth first receiving cavities 22 have a second end 25B that is located adjacent the opposite, second end face 18B. Adjacent the first end 23, the first receiving cavities have a greater depth than adjacent the second end. Consequently, liquid inserted adjacent the second end will flow in the direction of the first end of the relevant first receiving cavity, the advantages of which will be further discussed hereinbelow. The second holding means 7 comprise a plate part 26 provided with a number of series R and columns K of receiving cavities 27 having a relatively small content, for instance a few μl or ml. Each receiving cavity is substantially cup- shaped, as appears in particular from Figs. 3 and 4. The matrix plate-shaped second holding means 7 may be of a disposable type or may be reused after cleaning. The matrix plate 7 and receiving cavities 27 will be further described hereinbelow.

The analyzing means 8 are designed for analyzing the contents of at least one and preferably more, in particular all filled receiving cavities 27 in the matrix plate 7. For that purpose, the analyzing means 8 preferably comprise a so- called CCD camera ('charge-coupled device') whereby in each case an image can be taken of at least one row and/or column of receiving cavities 27, preferably one recording of all receiving cavities 27 at the same time, which recording or recordings can be analyzed in a manner to be further described hereinbelow.

Adjacent the supply means 2, pre-pickup means 28 are provided, comprising a suction needle 30 mounted on a movable arm 29, which needle can be moved into a container 3 for picking up an amount of sample 4, whereupon the filled needle 30 can be swiveled, by means of the arm 29, to a position above the pre-receiving cavity 14, whereupon the picked up sample can be dispensed from the needle 30 and received in said pre-receiving cavity 14. Next, the needle 30 can be swiveled away and rinsed, after which it is ready for picking up a new sample from a next container 3. This needle 30 will be further described in a preferred embodiment, with reference to Figs. 8A-D.

Next to the pre-pickup means 28, pre-dosing means 31 may be disposed, comprising a dosing needle 33 mounted on an arm 32 that is movable above the pre-receiving cavity 14, for inserting into the pre-receiving cavity 14, partially filled with sample, an amount of pre-treatment medium, for instance a solvent and/or anticoagulant for the blood sample if the latter was not added thereto already during blood-taking. By driving the drive means 15 in the manner described hereinabove, with the mixing block 9 performing a back-and- forth movement through said slight angle, a proper mixing of the sample with the pre-treatment medium is obtained in the pre-receiving cavity 14. The needle 33 is used only for the pre-treatment medium and hence need not be rinsed. Instead of the use of pre-dosing means 31, a or each pre-treatment medium may also be introduced into the pre- receiving cavity 14 via the needle 30, with direct rinsing of the needle 30 and the feed channel thereof.

Provided next to the pre-dosing means 31 are first pickup means 34, comprising pickup needles 36 mounted on a movable arm 35, the number of which pickup needles corresponds to the number of columns K of receiving cavities 27 on the second holding means 7. In the embodiment shown, five pickup needles 36 are provided, disposed in a row having a pattern corresponding to the pattern of the first receiving cavities 22 on the first holding means 6. By means of the pickup needles 36, five sub-samples can be taken from the pre-receiving cavity 14, which sub-samples can be dispensed at the first ends 23 of the first receiving cavities 22 (Fig. 5B) . The pickup needles 36 can then be swiveled away and cleaned, so that they are ready for use for a next sample. Provided next to the first pickup means 34 are first dosing means 37, comprising dosing needles 39 mounted on a movable arm 38 and disposed in a pattern corresponding to the position of the second ends 25A, 25B of the first receiving cavities 22. By means of the five dosing needles 39, a first treatment medium can be added to each sub-sample received in the relevant receiving cavity (Fig. 5A) , while the first treatment mediums introduced into the separate first receiving cavities 22 may differ from each other. Each first treatment medium may, for instance, contain one or more conjugates, whose purpose will be further described hereinbelow. Due to the arrangement of the dosing needles 39, these needles are prevented from contacting the sample and thus become contaminated.

Due to the drive of second drive means 40, the first holding means 6 can be driven in a back-and-forth movement around the axis 19, in accordance with the back-and- forth movement of the earlier described pre-holding means 5. Because of this back-and-forth, vibrating movement, a proper mixing is obtained of the sub-samples which have flowed together adjacent the first ends 23 of the first receiving cavities 22, and first treatment mediums added to those sub- samples. The first receiving cavities 22, downwardly inclined towards the first ends 23, have the advantage that at least the treatment medium can be introduced into the first receiving cavities 22 in a simple manner without the dosing needles 39 contacting the sub-samples. This prevents the necessity of cleaning the relevant dosing needles 39 after each dosing. The relatively deep first ends 23 offer the advantage that suction of the mixtures from the respective first receiving cavities 22 can thus be effected more simply. Provided next to the first dosing means 37 are second pickup means 41, comprising second pickup needles 43 mounted on a movable arm 42, which needles are arranged in a substantially straight line, while the second pickup needles 43 are distributed in accordance with the first ends 23 of the first receiving cavities 22. By means of the second pickup needles 43, a preferably exactly measured amount of mixture of sub-sample and relevant first treatment medium can be picked up from each first receiving cavity 22, and be transferred to the receiving cavities 27 on the second holding means 7 (Fig. 5B) . For this, the procedure is as follows.

The second pickup needles 43 filled with the amount of mixture of sub-sample and first treatment medium are moved with their open ends 44 to a position closely above the surface 45 of the plate-shaped second holding means 7, such that each second pickup needle 43 is located at the beginning of a column of receiving cavities 27. Next, the five second pickup needles 43 are moved in a direction parallel to the top face 45, parallel to the columns K, as shown in Figs. 6A and 6B by the arrow P. Due to the relatively small distance between the free end 44 of the needles 43 and the top face 45 of the matrix plate 7, the mixture of sub-sample and treatment mediums will be forced, through smearing, from the relevant needles 43 into the receiving cavity 27, such that the receiving cavity is filled completely, as shown in Fig. 4. The smearing movement of the needles 43, in particular said mixture, provides that all receiving cavities 27 are filled with a proper dose, without entailing the danger of air inclusion in the relevant receiving cavity 27, as a consequence of which one or more receiving cavities 27 could not or not completely be filled. Accordingly, relatively small receiving cavities can also be filled in a suitable manner, in spite of the liquid tension.

As is shown more particularly in Fig. 4, in at least a number of and preferably all receiving cavities 27, a second treatment medium is provided in the form of a coating 44 on which the mixture 45 of sub-sample and first treatment medium is provided. In a general sense, it could be stated that the coating 44 in question is selected for reaction with at least one constituent present in the mixture 45, which constituent may for instance be an antigen, an antibody, a serum or antiserum, an enzyme or the like, as is for instance customary in an Elisa test. The different receiving cavities 27 on the matrix plate 7 each have a specific coating 44, while receiving cavities 27 in the different columns K and/or rows R may have the same or different coatings 44. Accordingly, in each of the receiving cavities 27, a specific combination of a mixture of always the same sub-sample, a specific first treatment medium and a specific second treatment medium, i.e. coating 44, is formed. Thus, in each receiving cavity 27, a specific reaction will take place, indicative of the presence of a constituent that is to be specifically detected in the sub-sample. By way of illustration, on a matrix plate 7 having five columns K and five rows R, i.e. 25 receiving cavities 27, at least twenty- five different detections may in principle take place in one operation. Through specific combinations of active constituents, in particular different conjugates in each first treatment medium and adjustment thereto of the coating 44 in each of the receiving cavities 27 in the column K that is covered by the relevant dosing needle 43, different tests can be performed in each column K even more simply by means of the same mixture 45.

After filling of the different receiving cavities 27 with the relevant mixtures 45, the matrix plate is treated in the same known and customary manner, for instance through rinsing with a buffer and any further treatment mediums, after which the matrix plate 7 is exposed to the analyzing means 8. As stated, the analyzing means 8 preferably comprise a CCD camera, whereby a recording of all receiving cavities 27 can be made at once. The analyzing means 8 are connected to a computer 46 or a like calculating unit, in which the recording made can be analyzed. To that end, the computer 46 is provided with an algorithm for comparing the recording with standardized recordings stored in a databank in the computer 46. Each of the standardized recordings is made by means of samples of a known composition, so that with regard to these recordings it is known which constituents were present in the relevant sample and which were not. Hence, on the basis of the comparison made by the algorithm, it can at once be established which constituents were present in the sample 4, and optionally to what extent, which determination can be used for, for instance, determining a processing route of the human or animal from whom or which the relevant sample was taken, for instance in respect of the medicinal treatment, slaughter and the like. In fact, it is of course also possible to apply other types of analyzing means 8, known per se, while moreover, it is also possible to make one recording per receiving cavity 27 or per row R or column K of receiving cavities 27, for comparison by means of said algorithm. With the first drive means 15 and the second drive means 40 respectively, the block 9 and the block 17 can, in addition to said back-and-fort movement, also be rotated through an angle of 90° around the respective rotation axes 11 and 19. Thus, a next pre-receiving cavity 14 and a series of first receiving cavities 22 can in each case be brought into the overlying, horizontal position, while the pre- receiving cavity 14 or first receiving cavities 22 respectively, filled with the sample or mixture used, is/are rotated towards a vertical sideface. Disposed next to the block 9 is a first spray nozzle 47, and disposed next to the block 17 is a second spray nozzle 48, both spray nozzles being connected to pressure means 49 for spraying a cleaning medium whereby the pre-receiving cavity 14 and the first receiving cavities 22 respectively can be rinsed, against the sideface of the respective blocks 9, 17 that faces the relevant spray nozzles 47, 48, for cleaning it. Moreover, preferably, a third spray nozzle 49 is arranged adjacent the bottom side of the block 9, and a fourth spray nozzle 50 is arranged adjacent the bottom side of the block 17, which spray nozzles 49 and 50 are connected to second pressure means 51 for blowing air against the bottom face of the blocks 9 and 17, for drying the pre-receiving cavity 14 and first receiving cavities 22 respectively, located in the bottom face. This means that, during use, always a cleaned, dry pre-receiving cavity 14 or series of first receiving cavities 22 can be rotated into top face position for use, which can afterwards be cleaned and dried automatically, for which purpose the two blocks 9, 17 are always rotated in the same direction over 90°.

The apparatus 1 can be controlled by means of the computer 46. To that end, the drive means 15 and 40, drive means 52 for the supply means 2, the pre-pickup means 28, the pre-dosing means 31, the first pickup means 34, the first dosing means 37, the second pickup means 41, the first pressure means 49 and the second pressure means 51 are connected to the computer 46 designed for the control. Moreover, the treatment of the matrix plate 7 and the control of the analyzing means 8 can also be controlled by the computer 46.

Fig. 3 shows a cross section of a matrix plate 7, showing a receiving cavity 27 of each column K. Included between the columns K of receiving cavities 27 is a rib 53 whereby, during filling of the receiving cavity 27 by means of said smearing movement, mixture 45 of one of the columns K is prevented from mixing up with mixture on one of the other columns K. Fig. 3A shows an alternative embodiment where instead of the ribs 53, grooves 54 are provided, while in the embodiment shown in Fig. 3B, juxtaposed columns K of receiving cavities 27 are arranged at different levels. Also in the latter two embodiments, the mixture 45 in one column K is effectively prevented from mixing up with mixture 45 in an adjoining column K.

With an apparatus according to the invention, a large number of samples can be analyzed automatically at relatively high speed and in relatively short time, which renders this apparatus eminently suitable for analyzing samples in a (semi) continuous process.

In an apparatus 1 according to the invention, it is possible to insert the or each second treatment medium and/or further treatment mediums into the receiving cavities 27 on the matrix plate by further dosing means 27, not shown in the drawing. To that end, outlet openings may be provided in a pattern corresponding to the pattern of the receiving cavities 27, which outlet openings are individually controllable, for instance by means of the computer 46. In an alternative embodiment, the further dosing means comprise a series of further dosing needles movable over the matrix plate 7, at least the series and/or columns of receiving cavities 27, parallel to, at right angles to, or including a different angle with the direction P in Fig. 6B, while the relevant second or further treatment mediums can be inserted into the receiving cavities 27 preferably by means of the smearing movement described in respect of the second pickup means 41. Thus, any desired combination of mixture 45, second treatment medium 44 and any further treatment mediums can be obtained.

Fig. 7 schematically shows the arrangement of an advantageous embodiment of dosing means, in particular suitable for adding substrate to the cavities 27 in the matrix plate 7. Preferably, a number of such dosing devices are positioned in a matrix corresponding to the RxC matrix of the matrix plate 7. A dosing device 60 according to the invention comprises a chamber 61 having a bottom 62 and a top face 63. Extending through the bottom 62 is a hollow needle 64 having a beveled first end 65 located outside the chamber and a second end 66 located in the chamber at a distance B above the bottom 62. Moreover, a liquid discharge line 67 is passed through the bottom 62, whose feed opening 68, located in the chamber, lies at a distance S above the bottom 62, which distance S is at least less than the distance D and which may be 0. Extending through the top face 63 into the chamber are an air discharge line 69 and an air feed line 70, whose section is relatively large compared with the section of the air discharge line 69, the needle 64 and the liquid discharge line 67. In the air feed line 70, a valve 71 is provided, capable of closing or releasing the air feed line. On the liquid discharge line 67, shut-off means are provided, in particular pump means (not shown in the drawing) for discharging liquid through the liquid discharge line, as will be described in more detail hereinbelow. Connected to the air discharge line 69 are suction means (not shown in the drawing) , whose purpose will be described in more detail hereinbelow.

A device 60 according to Fig. 7 can be employed as follows .

The first end 65 of the needle 64 is positioned in a liquid to be picked up, in a container 72, as shown in Fig. 7A. Next, when valve 71 and liquid discharge line 67 are closed, air is drawn from the chamber 61 via the air discharge line 69, causing liquid to be drawn into the chamber via the needle 64, as shown in Figs. 7B and C. As the liquid discharge line 67 is closed, the liquid level in the chamber 61 can rise to above the second end 66 of the needle 64, as shown in Fig. 7D. Next, the air discharge means are switched off and the liquid discharge line 67 is released, allowing liquid to flow from the chamber 61, optionally drawn therefrom by appropriate pump means, as shown in Fig. 7E . The discharge of the liquid through the liquid discharge line 67 will be terminated when the liquid level in the chamber is flush with the feed opening 68, as shown in Fig. 7F. During these steps 7A-7F, the first end 65 of the needle 64 is held in the liquid in the container 72. Next, the first end 65 of the needle 64 is moved into a cavity 27 of the matrix plate 7 in such a manner that the bottom of the cavity 27 is not touched thereby, as shown in Fig. 7G. Next, the valve 71 is opened and relatively much air is introduced relatively quickly into the chamber 61 via the air feed line 70, while the air discharge line 69 and the liquid discharge line 67 are closed. The liquid that has stayed behind in the needle 64 during the discharge of the liquid from the chamber 61, as shown in Figs. 7E and F, is pressed from the needle 64 into the cavity 27 by the air pressure, as shown in Fig. 7H. Here, due to the beveled end 65 of the needle 64, the advantage is achieved that air is dispelled from the cavity 27, so that the formation of air bubbles in the cavity 27 is prevented. In this manner, splattering of the liquid, contamination of the environment of the cavity and the like are readily prevented, while the liquid can be introduced into the cavity 27 relatively quickly. When the cavity 27 is being filled completely, the needle 64 can be pulled upwards slightly, enabling the air from the chamber to flow away via the space between the opening in the hollow end 65 of the needle and the top face of the matrix plate 7 without influencing the liquid in the cavity. A device according to Fig. 7 has the advantage of enabling particularly accurate dosing in a particularly simple manner, as always the same amount of liquid will stay behind in the needle 64. Hence, this amount of liquid is the amount to be dosed, which will always be equal. Accordingly, the inside volume of the needle is the dosing quantity and can be exactly adjusted to the content of the cavity 27. Moreover, such device can readily be applied without requiring complicated systems of pistons, valves and the like. The advantage achieved by positioning a matrix of such dosing devices in accordance with the cavity 27 in the matrix plate 7, is that in one operation, a desired amount of substrate or like treatment medium can be accurately fed to each cavity 27.

Figs . 8A-D show a pickup and dosing device which is in particular suitable for picking up sample such as blood for supply to the pre-holding means 5 and, optionally, adding thereto a solvent or like pre-treatment medium. The dosing device will be described as pre-pickup and pre-dosing means. The pre-pickup means 28 comprise a hollow needle 30 as described earlier, provided with a beveled first open end 80. Provided around the free end of the needle 30 is a sleeve- shaped jacket 81, closed at its top end 82. The bottom end

83, which is for instance flush with the top side of the open end 80 of the needle 30, is open towards the bottom. The jacket 81 is preferably clamped on the needle, such that no air or liquid can pass upwards between the jacket 81 and the needle 30. Such a device can for instance be used as follows. The needle 30 with the jacket 31 is moved into the liquid 84 in a container 85, shown schematically in Fig. 8, where the liquid 84 is cross-hatched. Since the jacket 81 and the needle 30 are closed, air will be trapped in the needle and between the needle and the jacket 81 when introduced into the liquid. This prevents liquid from entering the space 86 enclosed between the needle 30 and the jacket 81 (Fig. 8B) . Next, an amount of liquid 84 is drawn into the needle (Fig. 8C) . Thereupon, the needle 30 together with the jacket 81 can be withdrawn from the liquid, while the liquid 84 stays behind in the needle 30 (Fig. 8D) . As no liquid has entered the space 86, contamination of the outside of the needle 30 is prevented in a simple manner, while, moreover, liquid 84 is prevented from being taken along on the outside of the needle and being dispensed unintentionally. Hence, with such a device, dosing can be effected particularly accurately. Indeed, the amount to be dosed is completely determined by the amount of liquid picked up in the needle 30. A further advantage is that such a dosing device can be maintained and cleaned in a particularly simple manner. For dispensing the liquid 84 from the needle 30, it is preferred that the jacket 81 be pulled away upwards over the needle 30, which prevents liquid that is possibly taken along on the jacket 81 from being delivered during dosing. For this purpose, the jacket 81 may for instance be bearing-mounted on the needle 30 so as to be slidable in a simple manner. Also, the jacket 81 may be designed for displacement relative to its top face. Many variations, which will readily occur to anyone skilled in the art, are possible hereto.

It will be directly understood by the skilled person that after substrate or a like treatment medium or mediums has been added to the cavities of the matrix plate 7 after the required steps like washing and the like, different further steps are to be taken, usually including washing steps, coloring steps, incubating steps and the like, before the matrix plate is supplied to the analyzing means. It will be understood that many variations are possible. For instance, the pre-holding block and/or the first holding means may be of different design, for instance having a triangular cross section at right angles to the rotation axis 11 or 14, or a polygonal cross section, while each side may contain one or more pre-receiving cavities or series of first receiving cavities. The number of series R and/or columns K on the second holding means 7 may be adjusted, for instance depending on the number of tests to be performed and the possible combinations of treatment mediums to be used, in particular mixtures of conjugates, while the number of first receiving cavities on the first holding means may be adjusted to the number of columns K. The means for cleaning and drying the different receiving cavities may be designed in a different, suitable manner, for instance as contact -cleaning means. Also, instead of the drive means, other means can be used for setting the blocks 9 and 17 into vibration, while, moreover, stirring means or the like may be used for mixing the samples and sub-samples with the different treatment mediums. Also, other pickup means and dosing means may be applied for transferring the samples, sub-samples and mixtures between the different phases in the treatment. Also, between the supply means and the pre-holding means, the pre-holding means and the first holding means, the first holding means and the second holding means and/or the second holding means and the analyzing means, further treatment means may be provided, for instance for adding further treatment mediums such as anticoagulants, solvents, buffer means, reagents and the like.

Claims

1. A method for serologically testing samples such as blood samples and the like, wherein at least one and preferably, a series of sub-samples are separated, wherein the at least one sub-sample is subjected to at least one serological test, wherein the at least one test is performed on-line.

2. A method according to claim 1, wherein a number of sub- samples are separated, said sub-samples each being mixed with at least a first treatment medium, after which at least a portion of each sub-sample is introduced into at least one of a column of receiving cavities in a matrix, to obtain a matrix of columns of receiving cavities filled with different sub-samples, wherein in at least each receiving cavity filled with sub-sample, a further treatment medium is mixed with the relevant sub-sample, after which the contents of at least a number of the filled receiving cavities are subjected to a suitable analyzing method.

3. A method according to claim 1 or 2 , wherein the sample is separated from an amount of sample-containing medium and is pretreated with at least a solvent or like pre-treatment medium, after which the sub-samples are separated.

4. A method according to any one of the preceding claims, wherein at least one of the treatment mediums comprises a mixture of at least two conjugates.

5. A method according to any one of the preceding claims, wherein in at least a number of the receiving cavities, prior to the insertion of a portion of the relevant sub-sample therein, the relevant further treatment medium is provided.

6. A method according to claim 5, wherein at least one further treatment medium or mixture of further treatment mediums is provided by coating the relevant receiving cavity.

7. A method according to any one of the preceding claims, wherein each sub-sample is received in or on a pickup part, preferably sucked up through a tubular end thereof, after which an open end of the pickup part is moved over the receiving cavities in the relevant column of the matrix, at such a small distance from the upper longitudinal edge thereof that the receiving cavities are filled with a measured amount of the relevant sub-sample, at least under the influence of cohesion, wherein any excess sub-sample is leveled off over the longitudinal edge of the relevant receiving cavity by the advancing pickup part .

8. A method according to any one of the preceding claims, wherein the receiving cavities are simultaneously subjected to a suitable analyzing method.

9. A method according to any one of the preceding claims, wherein as analyzing method a radiation measurement, in particular a light measurement is used, wherein by means of a receiver, an image of the matrix of receiving cavities is recorded, which image is compared with previously determined images, stored in a databank, of which images the desired measured values of the sample, at least of the constituents to be detected therein, for each of the relevant receiving cavities is known, wherein on the basis of this comparison, the desired measured value is determined for each of the relevant receiving cavities.

10. A method according to claim 9, wherein the databank is filled with a number of images taken of samples having a known composition, wherein the databank is supplemented with images of the samples to be examined, wherein by means of an algorithm in a calculating device, it is determined which of the measured images are relevant for incorporation into the databank, depending on at least a number of measured data of such samples, which measured data have been measured externally and inputted into the calculating unit.

11. A method according to any one of the preceding claims, wherein a number of separate samples are simultaneously treated in an apparatus .

12. An apparatus for on-line serologically testing samples such as blood samples and the like, comprising: first pickup means for picking up from a sample to be tested a number of sub-samples, preferably at least two sub- samples; first holding means for storing the sub-samples to be delivered therein by the first pickup means; first dosing means for adding a first treatment medium to each sub-sample in the first holding means; - first mixing means for mixing the or each sub-sample with the relevant first treatment medium added thereto; second pickup means for separately picking up each sub- sample from the first holding means; second holding means for separately storing the treated sub-samples to be delivered therein by the second pickup means ; said second holding means comprising a number of rows and columns of receiving cavities, preferably a matrix of receiving cavities; wherein the second pickup means are arranged for feeding the relevant, picked-up pretreated sub-samples into separate columns of receiving cavities and distributing them therein; wherein means are provided for adding a second treatment medium to the relevant part of the treated sub-sample in receiving cavities; and - means for analyzing the contents of at least a number and preferably all of the filled receiving cavities.

13. An apparatus according to claim 12, comprising: pre-pickup means for picking up a sample from a stock of samples ; - pre-holding means for sample to be delivered therein by the pre-pickup means; pre-dosing means for adding a pre-treatment medium to the sample in the pre-holding means; pre-mixing means for mixing the sample with the pre- treatment medium; wherein the first pickup means are arranged for picking up the sub-samples from the pre-holding means.

14. An apparatus according to claim 12 or 13, wherein a second treatment medium is provided at least in a number or preferably each of the receiving cavities, preferably prior to the introduction therein of the relevant part of a sub- sample .

15. An apparatus according to claim 14, wherein a number, preferably each of the receiving cavities to be filled are/is coated with at least a second treatment medium.

16. An apparatus according to any one of claims 12-15, wherein the receiving cavities are included in at least one plate part, preferably a matrix plate, wherein the second pickup means are arranged for passing a portion of the relevant pretreated sub-sample into the receiving cavities by a smearing movement over the or each surface of the relevant plate part .

17. An apparatus according to any one of claims 12-16, wherein the pre-holding means comprise at least one pre- mixing cavity, included in a pre-mixing block, wherein the pre-mixing means are arranged for selectively: - swiveling the pre-mixing block reciprocally over a relatively small first angle, or; tilting the pre-mixing block over a relatively great second angle in one direction, about a substantially horizontally arranged rotation axis.

18. An apparatus according to claim 17, wherein the pre- mixing block is provided with a pre-mixing cavity on at least two, preferably all sides, wherein the pre-pickup means are arranged for bringing the sample into the at least temporarily upwardly directed pre-mixing cavity, wherein cleaning means are provided for treating, in particular cleaning, the or each further pre-mixing cavity at least partially simultaneously.

19. An apparatus according to any one of claims 12-18, wherein the first holding means comprise a series of first mixing cavities, wherein the first mixing means comprise vibrating means for bringing the first mixing cavities into vibration.

20. An apparatus according to claim 19, wherein the first pickup means comprise a series of pickup elements disposed in a mutual position corresponding to the position of the series of first mixing cavities.

21. An apparatus according to claim 20, wherein the pickup elements are arranged in a row corresponding to the position of the receiving cavities in a row in the second holding means .

22. An apparatus according to claim 20 or 21, wherein a series of first mixing cavities are included on at least one side of a first mixing block, wherein the first mixing means are arranged for selectively: swiveling the first mixing block reciprocally over a relatively small first angle, or; - tilting the first mixing block over a relatively great second angle in one direction, about a substantially horizontally arranged rotation axis.

23. An apparatus according to claim 22, wherein the first mixing block is provided with a series of first mixing cavities on at least two and preferably all sides, wherein the first pickup means are arranged for bringing the sub- samples into the at least temporarily upwardly directed first mixing cavities, wherein cleaning means are provided for treating, in particular cleaning, the or each further series of first mixing cavities at least partially simultaneously.

24. An apparatus according to any one of claims 19-23, wherein the first mixing cavities have an inclined bottom face, and a content which is slightly greater than the volume of the sub-sample to be delivered therein, wherein the first dosing means are arranged for delivering, adjacent the highest end of the bottom face, each relevant first treatment medium, at horizontal and vertical distance from the treated sub-sample included in the relevant first mixing cavity.

25. A matrix plate, in particular suitable for use in a method according to any one of claims 1-11 or in an apparatus according to any one of claims 12-24, comprising a matrix of receiving cavities, wherein at least a number of receiving cavities are provided with a treatment medium for a sample or portion thereof to be introduced into the receiving cavity.

26. A matrix plate according to claim 25, wherein at least a number of receiving cavities are coated with a treatment medium.

27. A matrix plate according to claim 25 or 26, wherein in at least a number of columns of receiving cavities, at least one of the receiving cavities is provided with or preferably coated with a treatment medium different from the treatment medium in at least one of the other receiving cavities in the relevant column of receiving cavities.

28. An analyzing device for use in a method according to any one of claims 1-11 or in an apparatus according to any one of claims 12-24, comprising a measuring device for determining the radiation coming from, reflected by or absorbed by the contents of at least a number of receiving cavities, wherein means are provided for comparing the measured values with the corresponding measured values of known samples, which corresponding measured values are stored in a databank of the measuring device .

29. An analyzing device according to claim 28, wherein the measuring device comprises an algorithm for performing the comparison and, on the basis thereof, representing the serological test result of the relevant sample or at least a portion thereof, wherein the algorithm is arranged for adding relevant measured values found to the databank, the arrangement being such that the analyzing device is self- learning.

30. A method for processing meat products, wherein meat products to be processed, such as carcasses or parts thereof, are supplied in series, wherein by means of a method according to any one of claims 1-11 or in an apparatus according to any one of claims 12-24, and preferably utilizing an analyzing device according to claim 28 or 29, a blood sample is taken from at least a number of, and preferably from each of the supplied meat products, and is tested serologically, wherein at least partly on the basis of the measured values found by the analyzing method used, it is determined in what manner each of the meat products is further processed.

31. A meat-processing apparatus, comprising an apparatus according to any one of claims 13-24 and preferably an analyzing device according to claim 28 or 29, comprising means for supplying meat products in series, wherein the pre- pickup means are arranged for each time taking a sample of bodily fluid, preferably blood, from the supplied meat product, wherein the apparatus is arranged for serologically testing each of the samples taken, wherein discharge means for the meat products and control means for controlling the discharge means on the basis of the serological test results are provided.

32. A treatment medium for use in a method according to any one of claims 1-11 or in an apparatus according to any one of claims 12-24, wherein the treatment medium comprises at least two specific conjugates.