GB1565401A - Creation of Inventions and Patents with Artificial Intelligence

GB1565401A – Viewing housing for a diagnostic reagent holder and method
– Google Patents

GB1565401A – Viewing housing for a diagnostic reagent holder and method
– Google Patents
Viewing housing for a diagnostic reagent holder and method

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Publication number
GB1565401A

GB1565401A
GB44828/76A
GB4482876A
GB1565401A
GB 1565401 A
GB1565401 A
GB 1565401A
GB 44828/76 A
GB44828/76 A
GB 44828/76A
GB 4482876 A
GB4482876 A
GB 4482876A
GB 1565401 A
GB1565401 A
GB 1565401A
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GB
United Kingdom
Prior art keywords
holder
housing
reagent
film
viewing
Prior art date
1975-11-03
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)

Expired

Application number
GB44828/76A
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

International Diagnostic Technologies Inc

Original Assignee
International Diagnostic Technologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
1975-11-03
Filing date
1976-10-28
Publication date
1980-04-23

1976-10-28
Application filed by International Diagnostic Technologies Inc
filed
Critical
International Diagnostic Technologies Inc

1980-04-23
Publication of GB1565401A
publication
Critical
patent/GB1565401A/en

Status
Expired
legal-status
Critical
Current

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Classifications

G—PHYSICS

G01—MEASURING; TESTING

G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 – G01N31/00

G01N33/48—Biological material, e.g. blood, urine; Haemocytometers

G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

G01N33/54366—Apparatus specially adapted for solid-phase testing

Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC

Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10S435/00—Chemistry: molecular biology and microbiology

Y10S435/808—Optical sensing apparatus

Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC

Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10S436/00—Chemistry: analytical and immunological testing

Y10S436/80—Fluorescent dyes, e.g. rhodamine

Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC

Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10S436/00—Chemistry: analytical and immunological testing

Y10S436/805—Optical property

Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC

Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10S436/00—Chemistry: analytical and immunological testing

Y10S436/811—Test for named disease, body condition or organ function

Description

– 7 r 1 3
PATENT SPECIFICATION ( 21) Application No 44828/76 ( 22) Filed 28 Oct 1976 ( 31) Convention Application No 627941 ( 32) Filed 3 Nov 1975 in ( 33) United States of America (US) ( 44) Complete Specification published 23 April 1980 ( 51) INT CL 3 BOIL 9/00 GOIN 21/01 ( 52) Index at acceptance GIB CB ( 11) 1 565 401 ( 54) VIEWING HOUSING FOR A DIAGNOSTIC REAGENT HOLDER AND METHOD ( 71) We, INTERNATIONAL DIAGNOSTIC TECHNOLOGY, INC, a Corporation organised and existing under the laws of the State of California, United States of America, of 2551 Walsh Avenue, Santa Clara, State of California 95050, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in
and by the following statement: –
This invention relates to a viewing housing for a diagnostic reagent holder and to a method of determining substances using such a housing and reagent holders.
There are many techniques available for the detection of an unknown quantity of a biologically derived sample (e g, serum or urine).
During such techniques, a labelled substance which has reacted with the sample must be separated from the unreacted labelled substances which include free and non-specifically bound substance This separation in liquid form is known to be inefficient, unreliable, and tedious Many solutions have been proposed to solve this problem by the use of diagnostic reagents coated on a solid surface which combine with the labelled substance.
In one technique, reagents are coated upon plastic test tubes by physical adsorption of antibodies specific to the sample substance to be tested See, e g, articles by Catt et al in the Yournal of Biochemistry, 1966, Vol 100, page 31 c and in Science, Vol 158, page 1570, 1967 This technique is difficult to control because of the non-uniformity ofthe plastic surface and imprecision in the coating technique Furthermore, during washing to remove unreacted labelled substance, a relatively weak physical coating bond holding the antibodies can be disrupted resulting in their loss along with reacted labelled substance Also, this technique requires a separate procedure for the coating of each test tube This would be particularly time consuming, especially to insure reproducibility, if covalent attachment were employed to prevent the loss of diagnostic reagent In addition, such test tube coating does not lend itself to the precise viewing required in a solid front fluorometric system.
Furthermore, use of a test tube restricts covalent attachment to the material of construction used in making the tube.
Another solid surface approach is set forth in Bratu, Jr, et al U S patent 3,826,619 This system employs a physically adsorbed diagnostic reagent coated on the tip of a holder.
This tip is first fitted into a receptable for the sample and then into a receptacle for the labelled substance This system is subject to inaccuracies because of losses in rubbing of the unprotected tip against the close fitting receptacle Also, there is no ability to stir the tip in the receptacle leading to long incubation periods Additionally, this technique does not lend itself to reproducible mass production as each holder must be individually coated with diagnostic reagent Furthermore, it is undesirable for precise viewing.
According to the present invention there is provided a viewing housing for receiving a diagnostic reagent holder, said holder comprising an elongate shaft connected to a reagent containing surface, which housing comprises housing wall means including a front wall portion, means defining a reagent holder slot in said housing, stop means for limiting penetration of the reagent holder a predetermined distance into said reagent holder slot defining means such that said holder is repeatably positionable said predetermined distance into said reagent holder slot defining means, and a window opening through said front wall into said reagent holder slot defining means for viewing the reagent containing surface when the reagent holder contacts said stop means.
The invention further provides a combination comprising a viewing housing of the invention and a reagent holder disposed in said reagent holder slot defining means and in contact with said stop means and setting means with said reagent containing surface visible in said window.
One embodiment is a combination comprising a viewing housing of the invention and us 1565401 l an elongate diagnostic reagent holder having front and rear sides and comprising an elongate shaft, mounting means coupled to said shaft, self-supporting film bearing a diagnostic reagent firmly retained toward the end of said shaft by said mounting means on said front side, said mounting means comprising means defining a retaining groove at one end of said shaft, said film being retained in registry with said retaining groove.
A further embodiment is a combination comprising a viewing housing of the invention and an elongate diagnastic reagent holder having front and rear sides and comprising an elongate shaft, mounting means coupled to said shaft, self supporting film bearing a diagnostic reagent firmly retained toward the end of said shaft by said mounting means on said front side, and a locating means to assist registry of the holder with a cooperating portion of the housing.
Yet a further embodiment is a combination comprising a viewing housing of the invention and an elongate diagnostic reagent holder having front and rear sides and comprising an elongate shaft, mounting means coupled to said shaft, self-supporting film bearing a diagnostic reagent firmly retained toward the end of said shaft by said mounting means on said front side, and a pivot extension projecting from said holder at the film end and sufficiently aligned with said shaft to form a pivotal axis during stirring to facilitate liquid flow.
The invention also includes a combination comprising a viewing housing of the invention and a diagnostic reagent holder having front and rear sides and comprising an elongate shaft and a support surface adjacent one end of said shaft, self supporting film firmly adhered to the front side of said support surface, a diagnostic reagent coating affixed to said film, and protrusion means disposed externally adjacent the periphery of the film and projecting outwardly from the film a sufficient distance to protect the film from abrasion.
The invention additionally provides a method for the quantitative determination of a plurality of fluorescent or phosphorescent substances including a material derived from a biological fluid or tissue, wherein a sample of each substance is coated to a film on a respective one of a plurality of reagent holders as above, which method comprises the steps of:
a) inserting a first one of said reagent holders into the slot in a viewing housing of the invention until said film is precisely positioned adjacent the window in the housing, b) directing a beam of excitation light through said window against said film, c) receiving emitted light from said sample at a receiver of a fluorometer located at a predetermined distance from said window, d) measuring the intensity of the emitted light in said detector, and e) repeating steps a)-d) inclusive with subsequent reagent holders so that each film is positioned in said window at the same precise distance from said receiver during measurement.
It is the main object of the invention to provide a viewing housing particularly adapted 75 for precisely positioning a diagnostic reagent holder.
Our copending Application No 3503/79 (Serial No 1,565,402) divided from the present application, describes and claims diag 80 nostic reagent holders of the type referred to above.
The invention will now be further described and illustrated by reference to the accompanying drawings, in which: 85 Figure 1 is an exploded perspective view of the viewing housing of the present invention illustrating a diagnostic reagent holder and cuvette above their respective slots; Figure 2 is a top view partially broken 90 away of the housing of Figure 1; Figure 3 is an expanded cross-sectional view of a portion of the diagnostic reagent holder shown in Figure 1 taken along line 3-3; Figure 4 is a front view of a lower portion 95 of another diagnostic reagent holder; and Figure 5 is a cross-sectional view of the holder of Figure 4 taken along line 5-5.
Referring to Figures 1 and 3, a diagnostic reagent holder 10 is provided with a con 100 venient handle 11 at one end and mounting means comprising support surface 12 toward the other end interconnected by an elongate shaft 13 Support surface 12 is on the front or viewing side of the holder As illustrated, 105 handle 11 is formed in an enlarged rectangular shape of sufficient dimension transverse to shaft 13 for gripping either manually or by mechanical means This permits convenient stirring of a liquid with the holder and pro 110 vides support for a sample identification label.
Other handle shapes may be employed for this purpose Elongate shaft 13 serves to provide spacing between handle 11 and support surface 12 so that the holder may be stirred 115 by gripping handle 11 above a solution while support surface 12 is in the solution It facilitates rapid separation of solid and liquid phases It also permits precise positioning of support surface 12 in a viewing housing as 120 illustrated in Figure 1 with the support surface positioned deeply within the housing to shield it from ambient light The holder may be formed of any material relatively inert to the reactants such as moldable plastics 125 A self-supporting film comprising disc 16 is firmly adhered to the front side of support surface 12 by a suitable adhesive material or 1.56 ‘5401 plastic welding process A diagnostic reagent is carried by disc 16, preferably by covalent attachment, as described in detail hereinafter.
Support surface 12 and disc 16 are preferably of a circular shape to facilitate adhesion of the disc to the support surface without angular alignment.
Protrusion means comprising protective rim 14 surrounds a substantial portion of the periphery of support surface 12 and thus disc 16.
As illustrated, rim 14 extends around the entire periphery of the disc Rim 14 projects a sufficient distance outwardly from the exposed surface of disc 16 normal to the support surface to protect the disc from loss of material bound thereto by abrasion Other suitable protrusion means include a discontinuous rim with spaced elements or a number of circumferentially spaced points Such protrusion means also includes the inner wall of a flat front surface of the holder which surrounds a recessed support surface The outer surface of said protrusion means lies in a plane parallel to the face of disc 16 so that it may engage a cooperating surface of a housing of an optical instrument to precisely position disc 16 in the optical path.
A beveled edge 17 is provided on the lower end of the rear side of the holder to facilitate movement past a resiliently mounted projection for alignment in a slot of a viewing housing or the like.
A projection 18 is provided suitably also on the rear side of the holder opposite the center of the support surface to assist registry thereof with a corresponding housing recess.
In another alternative, the holder may be beveled parallel to the shaft to register with a corresponding projection on the slot.
A pivot extension comprising pointed projection 15 extends from the holder at the film end or bottom to form a resting point During stirring, the holder rests on point 15 in a liquid vessel, e g, test tube, to facilitate circulation of fluid around the bottom.
Disc 16 bears a diagnostic reagent capable of reacting with a sample substance For example, if the sample substance is an antigen, the diagnostic reagent may be an antibody specifically reactive with the antigen Thus, the diagnostic reagent comprises one of a pair or more of reactive substances The holder typically is employed in the testing of body fluids, such as serum, urine or other fluids, to ascertain the presence of pathogens or their toxins or to ascertain concentrations of other substances in the fluid The diagnostic reagent is a material which selectively or sterically fits with the mating sample substance Sample substances include drugs of abuse, such as morphine, methadone, cocaine, and barbiturates; drugs used for the control of certain chronic diseases or conditions such as digoxin (cardiac disorders), insulin (digitalis), and diphenylhydantoin (epilepsy); hormones such as thyroxine and triiodothyroxine; steroid hormones such as aldosterone, cortisol, testosterone, estriol and progesterone; peptide and protein hormones such as adrenocorticotropin, angiotensin, gastrin, chorionic gonadotropin, follicle stimulating hormone, growth hormone, luteinizing hormone, neurophysin, placental lactogen, and thyroid stimulating hormones, vitamins such as cyanocobalamin and folic acid; enzymes such as chymotrypsin, creatine phosphokinase, alkaline phosphatase, and lactic dehydrogenase; antigens such as carcinoembryonic antigen, hepatitis associated antigen and alpha fetoprotein; antibodies such as anti-toxoplasmosis antibody, anti-thyroid antibodies and anti-nuclear antibodies; cellular formed bodies such as bacteria, fungi, protozoa, erythrocytes and leucocytes; serum proteins such as fibrogens, anti-hemophilic factors, lipoproteins, immunoglobulins and thyroxine binding globulin; cellular degradation products such as, e g, myoglobins, bacterial toxins, and lyzozymal digests Other substances can be employed so long as they are detectable or rendered so as by direct labelling or through binding with labelled specific binding proteins, substances, inhibitors, enzymes, antigens or antibodies, and can be attached to a surface, either before or after they are directly or indirectly labelled.
Disc 16 is formed of an essentially nonswellable, continuous, impermeable material.
As defined herein, an impermeable film is one which will not permit the passage of liquid from one side to the other during the present process Although some penetration of liquid occurs, it is preferable that the film be suliciently non-porous to prevent significant penetration Use of this type of disc facilitates rapid washing of the surface after reaction with a labelled material to remove the background noise of unreacted labelled material In contrast, permeable porous surfaces require extensive washing The diagnostic reagent is preperably covalently attached to the surface of disc 16 Thus, disc 16 may comprise substrates such as a polyacrylic polyamide, cellulosic or other polymeric film depending upon the diagnostic reagent to be employed.
The structural strength of the holder required during handling, especially stirring, is not required for disc 16 Conversely, the chemical properties desirable for disc 16, e g, reactivity to form covalent attachments with diagnostic reagent is not necessary for holder Thus, it is sometimes advantageous to form the disc and holder of different materials.
If disc 16 is formed of polymeric materials which does not include within its matrix groups reactive with the diagnostic reagent, such reactive groups may be coupled thereto by known chemical reactions Groups of this type include amino groups, hydroxyl groups, mercapto groups, amido groups, and carboxyl groups Suitable attachment of diagnostic re1,565,401 1,565,401 agents to polymers with such groups coupled to them are set forth in Bennich et al U S.
patent 3,720,760.
Covalent attachment could be a time consuming difficult to reproduce operation in mass production if performed while disc 16 is secured to the support surface 12 It has been found desirable to first covalently attach the diagnostic reagent to a large number of the discs by agitating the same during reaction in a single vessel followed by adhering a disc to the support surface of each holder It is important to note that handling of the discs without protection by protective rim 14 and without the ability to freely move the same by holder 10 is not a problem at this stage.
Thus, abrasive contact with such discs to remove some diagnostic reagent would not result in inaccurate measurement procedures in carefully controlled manufacturing operations.
The discs are placed on the support surface prior to reaction with the labelled substance and sample substance as set forth hereinafter This protects the disc containing the sample and labelled substance from occasional abrasive contact which could result in the loss of signal detection and imprecision during analytical manipulations.
For simplicity of description, a typical fluorometrically labelled sandwich method will now be described.
In a first step, the diagnostic reagent (e g, antibody) is covalently attached to the disc in a manner as set forth above For mass production, it is convenient to form the discs by punching from a sheet of suitable material such as polyacrylic film Then, the discs are grafted with a spacer arm or coupling reagent A suitable reaction would include a large number of discs, e g, 100 or more, in a stirred reaction vessel Thereafter, the discs containing coupling reagents are reacted in a similar manner with a suitable antibody and washed and allowed to dry Such discs are then secured to the support surface 12 as with a pressure sensitive adhesion.
In the following step, the holder is gripped by the handle to place the diagnostic reagent bearing disc into a solution of a fluid containing sample substance reactive with the diagnostic reagent, e g, antigen If present, the antigen reacts and combines with the antibody on the surface during an incubation period.
It is advantageous to agitate the reactive substance during incubation This provides a significantly faster reaction time In addition, it has been found to increase the reproducibility of the experimental results, especially in short incubation periods Handle 11 provides a convenient means for mechanically or manually stirring of the solution.
After incubation with the sample serum containing antigen, holder 10 is simply removed from the solution and washed with a suitable solvent such as aqueous phosphate 65 buffer or distilled water.
In the following step, the labelled substance, suitably antibody labelled with a fluorochrome, radioactive substance, enzyme or phosphorescing substance, is contacted with 70 disc 16 and incubated for a sufficient time to complete reaction between the labelled antibody and antigen Again, it is advantageous to agitate the solution during incubation to decrease the reaction time and improve re 75 producibility of detection.
In the next step, the solution containing unbound labelled antibody is simply separated from the solid surface containing bound labelled antibody Thereafter, the reacted 80 solid surface is thoroughly washed to remove residual and non-specifically bound antibody which may remain on the disc The efficiency of this washing step is extremely important in obtaining accurate results Thus, the surface 85 is thoroughly washed with a suitable rinsing solution such as aqueous phosphate buffer or distilled water This illustrates the advantage of forming a firm covalent attachment of the diagnostic reagent with the disc to prevent 90 its loss with labelled antibody bound to it during this step.
After washing, the holder 10 is transported to a detection system for quantitative measurement In a particularly advantageous system, 95 the label is a fluorochrome and the detection system is a fluorometric system.
A description follows of a viewing housing suitable for incorporation in the above fluorometric system or other detection systems and 100 which is particularly adapted for precisely positioning of the above-described diagnostic reagent holder for optical detection.
Referring to Figures 1 and 2, a viewing housing 20 is illustrated which is particularly 105 adapted for receiving diagnostic reagent holder for viewing in a fluorometric system of the foregoing type Viewing housing 20 includes front wall 21, a top wall 22 and side walls 23 and 24 Housing 20 is slidably 110 mounted onto an L-shaped base member 26 which, in turn, is mounted in a stationary position in a detection assembly In the illustrated embodiment, housing 20 is provided with a longitudinal slot 27 extending parallel 115 to front wall 21 along the entire length of the housing Parallel detents 28 extending along the length of wall 27 are provided to mate with accommodating parallel grooves 29 in base 26 The cooperating detents and grooves 120 form track means for sliding horizontal movement of the viewing housing.
Means are providing for limiting lateral movement along she track means to define at least two precise predetermined lateral index 125 positions Such means comprises pairs of ball spring type set screws 35 mounted on the rear side of the housing projection into slot 27.
Screws 35 register with detents in the slot 51565401 at such index positions Pairs of spring mounted screws visible at the left side of the housing and also disposed at the right side are in registry with slot 27 to define a lateral path for the housing to ride upon A station selector handle 25 is provided to facilitate movement of housing 20 between the different lateral stations along track means.
In the illustrated embodiment, the housing includes at least two viewing slots, one for viewing holder 10 and another for viewing a cuvette In a fluorometric system, the viewing housing could be laterally moved so that the excitation and detector are rigidly mounted and housing 20 is moved into registry with the viewing slots A third slot, not shown, may be included for viewing another holder with a zero or standard reading.
Means defining a slot 31 is provided with an upper opening through top wall 22 The lower end of slot 31 is in registry with an opening through front wall 21 forming a window 32 into the slot Stop means is provided for limiting penetration of the reagent holder a predetermined distance along slot 31 In the illustrated embodiment, such stop means comprises the rounded lower portion 31 a of slot 31 A scrap drain opening 33 communicating with the interior of slot 31 and projecting out of the housing is provided to prevent build-up of scraps which might accumulate after long-term use In a fluorometric system as illustrated in Figure 2, light from a source travels along path A and contacts the disc on the support surface through window 32 and the emitted light is received along path B by a fluorescence detector.
Setting means is provided to urge the reagent holder against a surface of slot 31 to provide a precise positioning of support surface 12 in the viewing housing when the holder contacts the stop means In the illustrated embodiment, the setting means comprises a resiliently mounted projection in the form of spring mounted set screws 34 which projects toward front wall 21 Set screw 34 registers with projection 18 at the rear of holder 11 to precisely position support surface 12 in a predetermined fixed position adjacent window 32 with holder 10 in a viewing position.
Means defining a cuvette slot 36 is provided with an opening in front wall 21 Slot 36 is laterally spaced from slot 31 and includes a cuvette opening 36 a of expanded area in comparison to the remainder of the slot In the illustrated embodiment, the cuvette slot is of a square cross-section and is aligned in a 450 angle to front wall 21 Referring to Figure 2, when viewing housing 20 is employed in a fluorometric system, excitation light is supplied along path A and is reflected along path B for detection at a 90 angle.
The foregoing alignment facilitates this spacial relation Stop means comprising stop pin 37 is provided for cuvette slot 36 to position a cuvette at a precise elevation Alternatively, front surface fluorescence at other angles including 900 could also be measured using the system described in the aforementioned Harte patent application.
A fluid cuvette 38 of square cross-sectional area and transparent side walls fits precisely into cuvette slot 36 A top 39 is provided with an inlet tube 40 and an outlet tube 41 so that different diagnostic samples can be flowed into and out of the cuvette while it is in position.
Means is provided for sliding a light trap plate to cover whichever slot is not in use.
Such means comprises a recess in top wall 22 together with parallel dive tail slots 45 at each end of the recess A light trap plate 42 is slidably received in the recess and includes a pin 42 a to facilitate movement and stop pins 43 to define the extent of movement.
In operation of the diagnostic holder portion of the viewing housing, holder 10 is inserted into slot 31 until disc 16 is precisely positoned adjacent window 32 by means of set screws 34 registering with projection 18 in the back of support surface 12 In the fluorometric system, excitation light travels along path A to excite fluorescence on the fluorescent labelled disc that is received along path B to find a suitable fluorometer detector in which the intensity of emission is measured.
Alternatively, the above housing could be employed in other types of detection system such as radioimmunoassay using a geiger counter, or in a spectrophotometer If it is desired to analyze a fluid in cuvette 38, housing 20 is moved along its track laterally to align the cuvette with the detector.
The lower portion of another diagnostic reagent holder is illustrated in Figures 4 and Holder 46 includes a shaft 47 connected to mounting means comprising annular rim 48 with an internal retaining groove 49 extending the entire distance around the inner surface 50 of the rim A self-supporting film 51 bearing diagnostic reagent is secured into retaining groove 49 By forming disc 51 of a flexible film, the larger diameter disc may be flexed to slide into registry with groove 49.
Rim 48 also serves to protect disc 51 from abrasion A recess 52 is provided on the back side of shaft 48 to assist registry with a cooperating set screw 34 of housing slot 31.
Other recesses to assist registry include grooves in the holder adjacent the film and parallel to the shaft which align with corresponding projections in the sides of the viewing housing slot, e g, of wedge shape The remainder of holder 46 and the method of using it are the same as described with respect to holder 10.
In order to more clearly disclose the nature of the present invention, specific Examples of the practice of the invention are hereinafter 6 1 55 A O 6 given It should be understood, however, that this is done by way of example and is not intended to limit the scope of the invention.
Example 1 ( 1) Forming Covalent Bridges Acrylic discs, 1/4 inch in diameter, were punched from 6-mil thick polyacrylic acid film The discs were uniformly grafted with an amine bridge using a carbodiimide catalyzed nucleophile substitution reaction A typical reaction was carried out with stirring for two hours at room temperature using the following proportion of reactants:
discs 10 ml of p H 6 0 sodium phosphate 0 1 M buffer 0.1 g of 3,3 ‘ Iminobispropylamine 0.05 g of 1 cyclohexyl 3 ( 2 morpholinoethyl) carbodiimide methop toluenesulfonate After washing the discs, the amine bridge so formed was lengthened further by reaction with succinic anhydride, one end of which formed an amide linkage with the amine, the other end hydrolyzing to a carboxylic acid group on which protein can be immobilized.
A typical reaction was carried out with stirring for thirty minutes at room temperature using the following proportion of reactants:
100 discs mil of distilled water maintained at p H 6 0-7 0 by dropwise addition of 10 N sodium hydroxide 0.1 g of succinic anhydride added slowly over a twenty minute period ( 2) Covalent Attachment of Diagnostic Reagent After washing, the grafted discs were again reacted in a carbodiimide catalyzed reaction, this time with an antiserum A typical reaction, under the same conditions as above,was carried out using the following proportion of reactants:
adhesive The discs were attached to said surfaces by careful placement followed by a gentle pressure on the disc topsides to secure them to the holders for use in subsequent assays.
( 4) Reaction with Sample Substance Then one disc-containing reagent holder was inserted into a tube containing 0 5 ml of a p H 7 4 sodium phosphate 0 01 M buffer solution including 1 0 /I of human serum, and agitated gently at room temperature for minutes The reagent holder was then removed and washed with buffer solution.
( 5) Reaction with Labelled Substance After washing, the reagent holder was inserted into another tube containing 0 5 ml of a p H 7 4 sodium phosphate 0 01 M buffer solution including 5 0 /d of a commercial solution of fluorescein isothiocyanate conjugated goat immunoglobulin G derived from anti-human immunoglobulin G goat serum, and agitated gently by mechanical stirring at room temperature for 30 minutes The reagent was then removed and washed with buffer solution.
( 6) Measurement in Fluorometer After washing, the reagent was placed into the fluorometer holder and its fluorescent signal determined By comparison with fluorescent signals from known standard concentrations obtained similarly, as tabulated below, the sample concentration was found by interpolation to be 23 gg/ml Correcting for its dilution ( 1: 500), the original sample contained 11.5 mg/ml of immunoglobulin G.
Imnmunoglobulin G Concentration in Step 4 Ag/ml blank 1.1 28.5 56.9 sample Fluorescent Signal in Step 6 Arbitrary Units 72 357 763 321 discs 10 ml of p H 6 0 sodium phosphate 0 1 M buffer 0.05 ml of anti human immunoglobulin G goat serum 0.05 g of 1 cyclohexyl 3 ( 2 morpholineoethyl) carbodiimide methop toluenesulfonate.
( 3) Securing to Holder After washing, the antibody containing discs were allowed to air dry at room temperature Each of the support surfaces of diagnostic reagent holders of a type described herein were coated with one drop of an acrylic emulsion and allowed to dry to form a pressure sensitive Example 2
This Example illustrates the importance of precise repeatable positioning of the diagnostic 105 reagent holder with respect to the fluorometer.
Discs of 6-mil thickness were mounted on the support surface of the reagent holders as set forth in Example 1 The elevation of the discs were varied by increasing the thickness 110 of the adhesive layer separating the underside of the disc from the support surface The different discs were placed in the viewing housing of a type described above The optical system of the fluorometer was focused to give 115 a maximum signal at a 0 003-inch separation.
As this separation increased, fluorescent signal 1.565401 / 154 7 decreased as shown in the following series of measurements.
Approximate Fluorescent Signal Separation % of Maximum Distance, inches Series 1 Series 2 0.003 100 100 0.010 99 0.017 95 92 0 024 79 77

Claims (1)

WHAT WE CLAIM IS:-
1 A viewing housing for receiving a diagnostic reagent holder, said holder comprising an elongate shaft connected to a reagent containing surface, which housing comprises housing wall means including a front wall portion, means defining a reagent holder slot in said housing, stop means for limiting penetration of the reagent holder a predetermined distance into said reagent holder slot defining means such that said holder is repeatably positionable said predetermined distance into said reagent holder slot defining means, and a window opening through said front wall into said reagent holder slot defining means for viewing the reagent containing surface when the reagent holder contacts said stop means.
2 A viewing housing as claimed in claim 1 together with setting means for urging the reagent holder against a surface of said viewing housing when the holder contacts said stop means.
3 A viewing housing as claimed in claim 2 in which said setting means urges said reagent holder toward said slot defining means, said setting means comprising a resiliently mounted setting member projecting toward said reagent holder.
4 A viewing housing as claimed in any one of claims 1 to 3 together with means defining a cuvette slot, with a cuvette viewing opening in said front wall portion laterally spaced from said reagent holder slot defining means.
5 A viewing housing as claimed in any one of claims 1 to 4 together with a base member adapted for stationary mounting to a detection assembly, and cooperating track means mounted to said viewing housing and base member for sliding lateral movement of said viewing housing.
6 A viewing housing as claimed in claim together with means for limiting lateral movement along said track means to define at least two precise predetermined lateral positions.
7 A viewing housing as claimed in any one of claims 1 to 6 together with a source of light and a receiver of excited light both aligned with said window.
8 A viewing housing substantially as hereinbefore described with reference to and as illustrated in Figures 1 and 2 of the accompanying drawings.
9 A combination comprising a viewing housing as claimed in claim 2 or in any one of claims 3 to 7 when directly or indirectly dependent upon claim 2, and a reagent holder disposed in said reagent holder slot defining means and in contact with said stop means and setting means with said reagent containing surface visible in said window.
A combination comprising a viewing housing as claimed in any one of claims 1 to 8 and an elongate diagnostic reagent holder having front and rear sides and comprising an elongate shaft, mounting means coupled to said shaft, self-supporting film bearing a diagnostic reagent firmly retained toward the end of said shaft by said mounting means on said front side, said mounting means comprising means defining a retaining groove at one end of said shaft, said film being retained in registry with said retaining groove.
11 A combination comprising a viewing housing as claimed in any one of claims 1 to 8 and an elongate diagnostic reagent holder having front and rear sides and comprising an elongate shaft, mounting means coupled to said shaft, self-supporting film bearing a diagnostic reagent firmly retained toward the end of said shaft by said mounting means on said front side, and a locating means to assist registry of the holder with a cooperating portion of the housing.
12 A combination as claimed in claim 11 in which said locating means comprises a recess and said cooperating housing portion comprises a projection.
13 A combination as claimed in claim 11 in which said locating means comprises a projection and said cooperating housing portion comprises a recess.
14 A combination comprising a viewing housing as claimed in any one of claims 1 to 8 and an elongate diagnostic reagent holder having front and rear sides and comprising an elongate shaft, mounting means coupled to said shaft, self-supporting film bearing a diagnostic reagent firmly retained toward the end of said shaft by said mounting means on said front side, and a pivot extension projecting from said holder at the film end and sufficiently aligned with said shaft to form a pivotal axis during stirring to facilitate liquid flow.
A combination comprising a viewing housing as claimed in any one of claims 1 to 8 and a diagnostic reagent holder having front and rear sides and comprising an elongate shaft and a support surface adjacent one end of said shaft, self-supporting film firmly adhered to the front side of said support surface, a diagnostic reagent coating affixed to said film, and protrusion means disposed externally adjacent the periphery of the film and projecting outwardly from the film a 1,565,401 sufficient distance to protect the film from abrasion.
16 A combination as claimed in claim 15, in which said protrusion means comprises a protective rim around substantially all of the periphery of said film.
17 A combination as claimed in claim 15 or claim 16 in which said film is essentially impermeable to liquid.
18 A combination as claimed in any one of claims 15 to 17 in which said film comprises a disc bearing diagnostic reagent.
19 A combination as claimed in claim 18 in which said diagnostic reagent is covalently attached to an external surface of the disc.
A combination as claimed in any one of claims 15 to 19 in which said diagnostic reagent comprises antigen or antibody.
21 A combination as claimed in claim 20 in which said antigen or antibody bears a fluorescent or phosphorescent label.
22 A combination as claimed in any one of claims 15 to 21 also comprising an enlarged handle portion at the opposite end of the shaft from the film and of sufficient dimension transverse to the shafts to facilitate stirring a liquid with the holder and to support a sample identification label.
23 A combination of a viewing housing as claimed in any one of claims 1 to 7 and a diagnostic reagent holder substantially as hereinbefore described with reference to and as illustrated in Figure 3 or Figures 4 and 5 of the accompanying drawings.
24 A combination of a viewing housing and a diagnostic reagent holder substantially as hereinbefore described with reference to and as illustrated in Figure 1 of the accompanying drawings.
A method for the quantitative determination of a plurality of fluorescent or phosphorescent substances including a material derived from a biological fluid or tissue, wherein a sample of each substance is coated to a film on a respective one of a plurality of reagent holders as defined in any one of claims 10 to 23, which method comprises the steps of:
a) inserting a first one of said reagent holders into the slot in a viewing housing as claimed in any one of claims 1 to 8 until said film is precisely positioned adjacent the window in the housing, b) directing a beam of excitation light through said window against said film, c) receiving emitted light from said sample at a receiver of a fluorometer located at a predetermined distance from said window, d) measuring the intensity of the emitted light in said detector, and e) repeating steps a)-d) inclusive with subsequent reagent holders so that each film is positioned in said window at the same precise distance from said receiver during measurement.
26 A method as claimed in claim 25 and substantially as hereinbefore described.
INTERNATIONAL DIAGNOSTIC TECHNOLOGY, INC.
per: Boult, Wade & Tennant, 34, Cursitor Street, London, EC 4 A 1 PQ.
Chartered Patent Agents.
Printed for Her Majesty’s Stationery Office, by the Courier Press, Leamington Spa, 1980 Published by The Patent Office, 25 Southampton Buildings, London WC 2 A l AY, from which copies may be obtained.
8 1,565,401

GB44828/76A
1975-11-03
1976-10-28
Viewing housing for a diagnostic reagent holder and method

Expired

GB1565401A
(en)

Applications Claiming Priority (1)

Application Number
Priority Date
Filing Date
Title

US05/627,941

US3999948A
(en)

1975-11-03
1975-11-03
Diagnostic reagent holder and method

Publications (1)

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GB1565401A
true

GB1565401A
(en)

1980-04-23

Family
ID=24516751
Family Applications (1)

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Title
Priority Date
Filing Date

GB44828/76A
Expired

GB1565401A
(en)

1975-11-03
1976-10-28
Viewing housing for a diagnostic reagent holder and method

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US
(1)

US3999948A
(en)

JP
(2)

JPS5257317A
(en)

CA
(1)

CA1083729A
(en)

DE
(1)

DE2650106A1
(en)

FR
(1)

FR2329997A1
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GB
(1)

GB1565401A
(en)

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1975

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US
US05/627,941
patent/US3999948A/en
not_active
Expired – Lifetime

1976

1976-10-28
GB
GB44828/76A
patent/GB1565401A/en
not_active
Expired

1976-10-30
DE
DE19762650106
patent/DE2650106A1/en
active
Granted

1976-11-02
JP
JP51132243A
patent/JPS5257317A/en
active
Granted

1976-11-02
CA
CA264,659A
patent/CA1083729A/en
not_active
Expired

1976-11-02
FR
FR7633017A
patent/FR2329997A1/en
active
Granted

1979

1979-01-11
JP
JP54002118A
patent/JPS5914193B2/en
not_active
Expired

Also Published As

Publication number
Publication date

JPS5257317A
(en)

1977-05-11

JPS5533686A
(en)

1980-03-08

JPS5914193B2
(en)

1984-04-03

FR2329997A1
(en)

1977-05-27

US3999948A
(en)

1976-12-28

JPS5524067B2
(en)

1980-06-26

FR2329997B1
(en)

1982-05-07

DE2650106A1
(en)

1977-05-18

CA1083729A
(en)

1980-08-12

DE2650106C2
(en)

1989-06-29

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