CA1086524A - Nephelometer having means semiautomatically cancelling components from scattering by particles smaller or larger than those of interest - Google Patents
Nephelometer having means semiautomatically cancelling components from scattering by particles smaller or larger than those of interestInfo
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- CA1086524A CA1086524A CA342,031A CA342031A CA1086524A CA 1086524 A CA1086524 A CA 1086524A CA 342031 A CA342031 A CA 342031A CA 1086524 A CA1086524 A CA 1086524A
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Abstract
ABSTRACT OF THE DISCLOSURE
An improved nephelometer for immunochemical complex assay measures forward light scatter in samples. The angle of forward scatter, about 30°, is small enough to result in a large amount of forward scatter from the immunochemical complex particles which are to be assayed, whose size is of the order of the wave length of the light used in the optical system. Forward scattering from smaller particles, such as from molecules of buffer, antibody and serum, is constant during the course of a test, and is compensated for by the use of subtraction circuits which are readily and semiautomatically adjusted to subtract proper values, in accordance with the readings taken on standard or "blank" samples of buffer, antibody and serum. Forward scattering from large particles, such as dust, is variable, and results in fluctuating signals, which are electrically processed to ignore the spurious peaks. The results of a test are displayed on a digital read out meter. Also described is a method of and protocol for immunochemical assay, whereby the amount of antigen originally present in a sample is determined by adding a known amount of antibody, and by assaying the "blank" component ingredients and the resulting mixture.
An improved nephelometer for immunochemical complex assay measures forward light scatter in samples. The angle of forward scatter, about 30°, is small enough to result in a large amount of forward scatter from the immunochemical complex particles which are to be assayed, whose size is of the order of the wave length of the light used in the optical system. Forward scattering from smaller particles, such as from molecules of buffer, antibody and serum, is constant during the course of a test, and is compensated for by the use of subtraction circuits which are readily and semiautomatically adjusted to subtract proper values, in accordance with the readings taken on standard or "blank" samples of buffer, antibody and serum. Forward scattering from large particles, such as dust, is variable, and results in fluctuating signals, which are electrically processed to ignore the spurious peaks. The results of a test are displayed on a digital read out meter. Also described is a method of and protocol for immunochemical assay, whereby the amount of antigen originally present in a sample is determined by adding a known amount of antibody, and by assaying the "blank" component ingredients and the resulting mixture.
Description
2~ -BRIEF ~UMMAR~ OF INVENTION
This invention relates to the use of nephelometry to assay the amount of immuno-chemical complex present in a sample.
The invention is based on the fact that in an immuno-chemical complex assay, the various ingredients present have diverse sizes. The particles, including dissolved macromolecules, in buffer, antibody and serum are much smaller than the particles of immuno-chemical complex, while fortuitous particles of dust are generally much larger. As a result of this difference in size and the difference between the large concentration of buffer, antibody and serum present and the small concentration of dust present, it follows that the scatter due to the dust particles fluctuates while the scatter due to all other components is reasonably steady.
Thus in accordance with this invention there is provided in a system for measuring the concentration of medium sized particles in a mixture with both larger and smaller particles, said medium and smaller particles being substantially constant in a macroscopic field of view and said larger particles fluctuating in said field of view, the following combination:
means for directing a beam of electromagnetic radiation through said mixture of particles, means for establishing said field of view, and for sensing and measuring the scatter, in said field of view, from said beam along a direction off-axis to the beam, said scatter being a measure of the combined concentration of all particles present in said mixture, means for suppressing the fluctuatiOn in said measurement, due to the fluctuation of the larger sized particles, by clipping out the fluctuating peaks of the measurement during a period of time, thereby giving a steady ..
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measurement determined by the minimum measurement over said perlod of time, said steady measurement being indicative of the combined concentration o~ medium and small sized particles in said mixture, ;
and means for subtracting from said steady measurement an amount known to represent the concentration of the smaller particles, thereby giving a measurement indicative of the concentration of only said medium sized particles in said mixture.
As a further embodiment of the invention the com-bination described above may also include means for sequentially measuring the concentration of different ones of said smaller ;
sized particles by sequentially sensing scatter from samples of said smaller sized particles, said samples of smaller sized particles being related, in concentration, to the corresponding concentrations in said mixtures of said particles, means sequentially and automatically storing the measurements of concentration of said different smaller sized particles, said storing occurring at the time said sequential measurements are made, and means for utilizing said stored measurements in said means for subtracting.
Further embodiments of the invention comprise a nephelometer and its use for detecting and measuring the quantity of antigens present in a sample as more completely described hereinafter.
The nephelometer itself is characterized by these ;
features:
The optical system uses an arrangement which accepts samples in test tubes and accurately measures forward scatter o~
-a small volume of liquid, spaced within the walls of the test tube.
The design of the Gptical system is such that specular reflection of the incident beam on th~ test tube walls does not interfere with cm/~ ~ 3 ~
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measurement of the scatter, and thus cornmercial test tubes are satisfactory for use as test cells.
The angle of forward scatter is chosen to be about 30 from the illuminatiny beam within said small volume, as it has been found that for a broad range of angles centered on this value, the ratio of desired scatteriny, from the immuno-chemical complex particles of interest to the scattering, not of interest, by the larger and smaller particles that are present in thé assay, is considerably higher than at other scattering angles.
The scatter due to large particles, such as dust, is irregular and is ignored by the electronic circuitry, which measures the minimum value of sca'cter signal over a period of time.
The value of scatter due to particles smaller than those of interest is subtracted from the total scatter to determine the amount of scatter from particles of interest. This subtraction is done semiautomatically by the instrument, in accordance with readings made on the standard or "blank" solutions o the particles ~;
smaller than those o~ interest.
There is described below a certain protocol in ~ ~
accordance with which =he assay 's per/ormed ~
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BRIF,F Dl;SCRi PTION OF VI~;WS OF DR~WING
__ Figure 1 is a vertical cross-sectional view taken through the optical portion of an improved immunoassay nephelo-meter constructed in accordance with the present invention.
Figure 2 is an enlarged horizontal cross-sectional view taken substantially on the line 2-2 of Figure 1.
Figure 3 is an enlarged fragmentary vertical cross- -sectional view of the light-scattering portion of the nephelo- ;~
meter of Figure 1.
Figure 4 is a perspective diagram, corresponding to Figure 3, illustrating how the photomultiplier field of view is restricted by field stops so as to permit it to observe forward scatter from only a limited portion of the sample within the test tube.
Figure 5 is a simplified block diagram of the electrical signal processing and control circuitry of the nephelometer. -Figure 6 is a block diagram of another embodiment of immunochemical complex nephelometry assay system.
Figure 7 is a diagram of the electrical control and measurement sections of the nephelometer, showing the relation-ship of the parts to the front panel controls.
Figure 8 is a perspective view of a preferred em-bodiment of the nephelometer, showing the front panel controls and displays and showing the optical testing station.
Figures 9A and 9B are a schematic of the circuit used in the nephelometer of Figure 8.
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~CKGROUND OF INVENTIOM
_ In the bioloyical laboratory nephelomctry is a standard tool for quantitatively measuring the amount of certain biologicals in a liquid sample. The measurement is made by directing a beam of light through the liquid, and determining the amount of light which is scattered at different angles. The amount which is sca-ttered depends on the size of the scattering particles, their concentration, their shape, the wavelength of the light used, -the refraction indexes of the particles and of the medium in which they are suspended, and the angle at which the scatter is measured. There is a vast body of learning which enables a determination of the concentration to ~e made from such observations, if the dis-tribution of particle sizes is ~nown, but there are practical -difficulties.
For example, in the particular instance which led to the present invention, it was desired to measure the amount of an immunochemical complex in a sample.-From filtration tests made to determine the approxi-mate size of the particles giving rise to the desired and undesired scattering signals, respectively, it was determined that the desired immunochemical complex will pass through a 0.4 micron filter but is stopped by a 0.2 micron filter. Its .. . .
size is therefore, roughly, about 300 nanometers.
The angle chosen for measurement of the scattered -light strongly influences the magnitude of the measurement.
A small forward angle increases the amount of scattered light, as is obvious when considering the appearance of lights at night throllgh a fog. Furthermore, the larger and .
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medium sized particles are relatively more effective in seattering light at small forward anyles than aré the small particles. ~hus, in order to minimize the contribution of small sized particlas, small forward angles are desirable.
~lowever, strong scattering is also obtained from large dust particles which are unavoidably present in the samples. Thus, to minimize the contribution of large sized particles, larger angles of forward scatter should be used.
In making the measurement, the invention seizes on the fact that the particles which are of interest are the largest of the particles present in a constant macroscopic manner. Larger particles, namely, dust, dance around in the so.lution and their scattering is not constant because the dust particles, relatively few in number from a statistical point of view, are not constant in the field of the light beam. .:
Aecordingly, the invention measures the desired eomponent by subtraeting from the observed value the constant eontribution from particles smaller than those of interest while eleetrically suppressing the fluetuating contribution from dust, .
so that it is not included in the measurement.
It has been found that there is a broad range of forward .
seatter angles, eentered at about 30, where the angle chosen inereases the ratio of scatter by medium and large sized partieles to scatter by small sized partieles, without also inereaslng the seatter from the large sized particles to sueh an extent as to o.verload the ability of the electrical system to suppress the ~ fluetuating eomponent representing the large sized partieles.
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D~TAII,ED DESCRIPTION
Referring to the drawings, 11 generally designates the optical p~rtion of a nephelome~er in accordance with the present invention. The nephelometer 11 comprises a housing 12 in which is suitably mounted a horizontally-directed laser unit 13, for exam~le, a Model 155 laser unit, manufactured by Spectro-physics, Mountain View, California, which yenerates a 632.8 nm tnanometer) laser beam, shown at 14.
Housing 12 is provided with a vertical sample-receiving chamber 15 having a relatively thick right side wall 16, as viewed in Figure 1, front and rear walls 17 and 18, a left side wall 19, and a bottom wall 20. Chamber 15 is provided with a removable top cover 21 having a peripheral flange 22.
Right side wall 16 is formed.with an inwardly facing vertical V-groove 23 adapted to be engaged by a conventional vertically positioned test tube 24. A verti.cal pressure block 25 formed with an inwardly facing vertlcal V-groove 26 is :~--slidably positioned in the left side chamber 15, as viewed in .
Figure 1, and is provided with a pair of vertical bowed wire : springs 27,27 secured symmetrically to the left side face of ; the block so as to bear against chamber wall 19 and cause the V-grooved block 25 to exert positioning spring ~orce against the test tube 24 to hold the test tube firmly against V-groove : 23 of wall 16, as shown in Figure 2.
Wall 19 and block 25 are formed with respective laser beam apertures 28 and 29 aligned with beam 14. Wall 16 is formed with a vertically extending recess 30 including a light trap recess 31 aligned with beam 14 and lined with suitable :
light-absorbing material. Recess 30 communicates with a 45- ~ i , , .. .
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inclined li~ht passage 32 formed in wall 16 and provided at its opposite ends ~ith collimating field stops 33 and 3 having 2.8 divergence round field stops as above described aligned at ~5 with respect to beam 14 and aimed, or a conventional 12 x 75 mm test tube 24 at a point on the wall of the test tube about 3.07mm above the beam center line.
Field stop 34 opens into a light tube 35 leading to a photomultiplier tube 36 mounted in a chamber 37 provided therefor on housing 12, as shown in Figure 1.
In some conventional nephelometers utilizing test tubes as test cells, the incident beam and the observed scatter lie in a plane perpendicular to the axis of the test tube.
In such an arrangement, incident rays are multiply reflected from the sides of the test tube in the same plane as the observed scatter and are picked up without discrimination.
One of the features of the instant nephelometer is the placement of the test tube axis in the same plane as that of the incident and scattered rays, thus avoiding the spurious signal due to multiple reflections from the sides of the test tube. These spurious signals are not reproduceable, using different test tubes. The multiple reflections referred to are those at the glass-air interface, and they exist in the absence of imperfectlons, but are ;~
widely different with r~ifferent test tubes because of the large number of reflections involved.
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As can be seen from.Figure 4, the laser beam strongly illuminates a section of the test tube 24 and its contents between entrance 24A and exit 24D. At these two spo-ts, the unavoidable surface deposits and artifacts usually found on and in the glass or plastic of laboratory test tubes or cuvettes and the irregulari-ties of the tube-liquid interface and tube-air interface cause a strong forward scatter, which is unrelated to the forward scatter which it is desired to measure. In order to eliminate this undesired forward scatter from the measurement, field stops.33 and 34 coopera-te to define :~
a family of extreme rays, two of which, 63A and 63B, are -shown. These two extreme rays lie in a vertical plane containing the vertical diameter of each of the field ~.
stops 33 and 34~ These two extreme rays, which are shown as breaking at the rear surface of the test tube, define a part of the front and rear limits of view 24B and 24C.
The portion of the test tube volume between these two ~ ~
limits of view and also illuminated by the laser beam, ;
is accessable to view by the photomultiplier tube 36. :
This tube has an entrance aperture 36B which is larger than, and surrounds the exit aperture 36A of the field -stops 33 and 34.
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l'he field stops _3 and 34 are mounted vertically and therefore define a field of view whose right cross section is elliptical rather than round. This is a matter of convenience .in using round field stops and vertical mountings.
Although the effectively used portion of the tube 24 appears in Fiqure 4 to extend almost from inner tube wall to inner tube wall, the showing is exaygerated to render details visible. In our pre~erred embodiments, we have used an.effective portion about 1 millimeter in major dimension, near the center of a 10 millimeter diameter test tube. This disproportion in size increases the accuracy of measurement.
Designated at 38 (Figure 2) is a safety shutter plate which is slidably engaged against left wall 19 and which has a bottom flange 39 through which is slidably received a headed vertical pin 40 rigidly secured to the bottom wall of housing 12~
A coiled biasing spring 41 surrounds the lower portion of the pin 40 and bears between flange 39 and the housing bottom wall. The .
top end of plate 38 extends through a guide slot 42 in the top wall of housing 12 located beneath the peripheral flange 22 of top cover 21. Shutter plate 38 has a light aperture ~3 which ~ is moved into registration with wall aperture 28 when top cover 21 is seated in closed position ovçr the top end of chamber 15, as shown in Figure i. When the cover 21 is removed, spring 41 ~ :
elevates the shutter plate 38 into blan~ing position covering aperture 28. This insures against dangerous laser flash hazards which may be present when an operator looks downwardly into the test tube 24 with cover 21 removed.
As above e~plained, and as shown in Figures 3 and 4, there is refraction at the wall of test tube 24, so that the .
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exit a~le of the sc~tter beam, sho~l at 63, is 45 to the incid~nt laser be~m 14, whereas inside the test tube the scatter angle is 31.6 to the incident laser beam. I~le angle 45 is a conveni~nt manufacturing angle, .
and the cons~quential angle of 31.6 has been found to give a high ratio of scattering from immuno-chemical complex particles to scattering from smaller particles, without overloading the electrical processing circuit (which will be explain~d belcw) with excessive fluctuating noise signals arrising from the presence of l æge particles, such as dust. It will also be seen that the angled scatter beam seen by the photGmultiplier tube 36 is in a vertical plain containing the axis of the test tube,thereby eliminating artifacts due to internal reflections in the test tube, as absve explained. -Test tubes satisfactory for use ~ith the invention are from commercial stock. For example, ordinary Kimble test tubes, 10 x 75 mm æe satisfactory, unless they have defects in the wall obvious to brlef visual inspection.
In operatiol~, particles of approximately 0.3 micron size in the liquid in test tube 24 scatter the laser beam 14 strongly along the optical path defined by the field stops 33,34 and generate corresponding signals in the photomwltiplier tube 36, so that these signals can be used to measure the a~ount of antigen originally present in the test tube, after a kncwn amount of antibody material is added to the liquid contained in the test tube..................................................... . ?
At low antigen levels and relatively low forward scattering angles, such as the ar~?le employed herein (approximately 31.6) the influence of dust particles in the test tubet and similar artifacts, becomes quite Lmportant and presents.a source of serious error. The drawing, Fi~. 5, illustrates in block fonm a signal-handling circuit which discriminates against sporadic positiv~ spike effects caused by such dust particles or other artifacts. . . . .. ..
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A steady offset voltage greater than the maximum photomultiplier signal is applied at 45 to the other input of inverter/and adder 44B. The input signal at 47 is inverted in the inverter 44A, as shown.at 72, and the inverted peaks are shown at 73. The steady po'sitive input voltage applied at 45 is added in the adder 44B to the signal 72, and the resultant positive output signal at 46, shown at 74, has positive maxima corresponding to the original minimum values 75 of the input signal applied at 47.
The output signal at 46 is'passed through a timer switch element 76, an electronic switch 48, a diode 49 and an operational amplifier 50, providing an output signal at 52 in the form of a positive peak when the variable input at 47 'is a minimum. The greatest positive peak signal during the sampling period reaching operational ampli~ier 50 is held in capac'itor 77 to develop a steady comparison signal, available at 52, which is applied at 78 to one input of a comparator 53. The output s'ignal 74 from device 44 is applied (with timed switch element 76 closed) to the other input 79 of comparator 5~.
.Switch 48 will close initially at the init'ial value of curve 74~ Capacitor 77 will charge to this value, at which point switch 48 will open by the action of comparator 53.
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The value at 52 will be hel~l until ~he curve 7~ amplitude exceeds the initial stored highest point value at 52, at which point switch 48 will again close. The value at 52 will now assymptoti-cally track tincrease) ~he relatively stable positive maximum values of curve 7~, giving a new stored value at 52. Curve 74 will continue to establish such new stored values (with each increase). The comparator ignores the effects of minima caused by artifacts ~suchas Ininima 80). At any time that the amplitude of curve 74 ~oes below the tracked stored value at 52, switch 48 opens, and the signal at 46 is not forwarded to the inte-grating capacitor 77 for storage. Nor can the positive signal stored in capacitor 77 be lost unless reset switch 88 is closed.
These positive signals cannot be lost through diode 49 when the signal at output 46 ls more negative (as at 80) than the value stored in integrating capacitor 77 because of the poling of diode 49, which permits positive charge to flow only from left to right.
The positive signals cannot be lost through operational amplifier 50 because the input impedance of the operational amplifier is enormous, amounting effectively to an open circuit.
Thus, electronic switch 48 opens when the signal at 78 is greater than the signal at 79. The maxima of signal 7~ will, however, be stored in integrating capacitor 77. The stored signal at 52 is thus compared with the instantaneous signal at 46 in the comparator 53, whose output controls switch 48 so that switch 48 is held closed only when the variable input at 47 is higher than a previously stored value. Therefore, switch 48 is normally closed (no signal at 52) and opens when the signaL 74 goes below its prior stored value.
The desired positive antigen scatter signal at 52 is summed in an algebraic summing amplifier 54 with a suitable negative - . .
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offsct signal applied at 8], with a negative antibody blank signal applied at 82, adjusted in a manner presently to be .¦
described, and with a negative stored serum ~antigen) blank .
signal from an integrator 83, applied at 84. The output signal from amplifier 54 i.s delivered through a timed switch element ~5 to a digital display device 56. The disylay provided by device 56 is for a selected period of opera~ion of the algebraic summing amplifier 54, con~rolled by a manually activated computing timer 8fi. Timer 86 is designed to provide a signal sampling period of at least several seconds in order to discriminate adequately against large dust particles or similar artifacts.
When timer 86 is activated by its starting switch, shown at 87, it discharges the signal storage capacitor 77 by momentarily closing a reset switch 88 connected across the ~:
capacitor, closes the timer switch element 76 and opens the ; switch element 85. This provides several seconds for the ac-cumulation of the antigen scatter signal in capacitor 77. At the end of the timed period, switch element 76 opens and closes, producing thè digital display.in device 56.
The negative antibody blank adjustment signal at 82 is obtained from a potentiometer 90 connected to a suitable voltage source. The potentiometer is adjusted ~with its control - switch 91 closed) to provide a negative signal at 82 such as. to give a zero readout on device 56 in a prior test on a blank reference sample of antibody material.
The negative antigen (serum) blank signal at 84 com-prises a compensation signal obtained from the output o~ algebraic summing amplifier 54, with the antibody blank switch 91 open, in a prior test on a plain antigen reference sample before antibody . ' ' ~'' '-' .':', -' . ' ' . .
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material is added~ This ar,tigen tserum) blank signal is stored in integrat:or 83 by activating a "set" switch 93 during such a prior test. Thc stored negative antigen blank signal is then ' available to supply the required compensating negative blank antigen (serum) signal at 84 during the test on the final mix-ture above described.
It will therefore be seen that by subtracting the preset antibody blank signal at 82 and the stored antigen blank signal at 84 from the main test signal at 52, the required discrimination against other particles, such as free antibody and other particles in the antibody blank and free antigen and other particles in the serum blank, is accomplished, and the digital display of the immunocomplex particles is not affected by the presence of said other particles in the final test mixture. This digital display can ther,efore be used to provide an accurate indication of the amount of antigen originally present in a sample after the sample has been exposed to a known quantity of antibody reagent.
The operation of the system of Figure 5 can be under- ,~
stood more easily if it is compared to that of the simpler em-bodiment of Figure 6. These two embodiments differ in that the system of Figure 6 is a four-channel system in which four optical measurements are simultaneously made and simultaneously processed to produce the desired measurement display at 56a. In the system of Figure 5 the corresponding four optical measurements are 'sequentially made, the, results of the first three tests are stored, , and these stored measurements are combined with the fourth measure-ment to give a measurement indication at digital display S6a.
In Figure 6 there are four optical test stations lla to lld. At station llat a forward scatter measurement is made of -.: ' ' ' , ~ , , Z4~
the bufer sol~tion, ~Ihich is ~vailablc in such purity as to contain negligible dust. EaCh of the other biological reagents which must be used, the antibody and the serum, which are measured at stations llb and llc, necessarily have dust in them because of the manner in which they are obtained. At the fourth station there is measured a solution which contains as ingredients both the antibody and the serum sample, which react to produce the immunochemical complex whose concentration is to be indicated at 56a.
The signal at line 101 is one whlch corresponds sub-stantially only to the scattering property of the pure buffer.
Since buffer is in the mixtures at stations llb to lld, and the buffer signal is similarly subtracted from other signals at difference amplifiers 108 to 110, the buffer signal, in effect, ` sets the zero operating point or datum reference or the instrument.
The signals at lines 102 to 104 are irregularly affecte~
by dust, and the dust signals are eliminated by means of the .,; . .
minimum point memory circuits 105 to 107. These circuits correspond to the minimum point memory circuit of Figure 5, comprising inte-grator capacitor 77, diode 49, electronic switch ~8 and comparator 53.
The signals from differential amplifiers 108 to 110, when comb1ned in proper sense in algebraic sum amplifier 54a, produce the immunochemical complex signal which is fed to measurement display . 56a.
It will be seen, by tracing the signals in Figure 6, that each of the undesired signals is eliminated from the output of algebraic sum amplifier 54a, either by cancellation in minimum ; point memories 105 to 107 or by subtraction in differential amplifiers 108 to 110 or algebraic sum amplifier 5~a.
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Fi~urc 7 is a simplified block diagram of the invention showing how the objec~ives ar~ accomplished. No attempt has been made to illustratc ~he actual circuits, but to show Eunctiona~
characteristics. Probably the best way to describe the operation of the system is to follow the protocol and explain what ha~pens as the operator adjusts the knobs or pushes the di~ferent buttons.
For this purpose the diagram shows, adjacent the appropriate place in the circuit, the label which is affixed to the front panel con-trol which is manipulated by the operator during the test.
A significant point that must be remembered for proper understanding is that there are four SPECIMEN SWITCH positions, BUFFER BLANK, ANTIBODY BLANK, SAMPLE BLANK, and SAMPLE READ, which are mutually exclusive.
~ Protocol A. Rfter a 30 minute warm-up, turn HIGH VOLTAGE on. This con~
nects negative voltage to the cathode of the photomultlpliex tube.
B. Lift sample station cover. PHOTOMETER BLANK switch to MEDIUM.
SENSITIVITY switch to X3. Adjust analog meter to zero u~ing the PHOTOMETER BLANK fine controls~ This step establish~s electronic "zero". -C. Put a tube containing the highest "reference" concentration of antigen/antibody into the sample station and adjust the . . .
sensitivity controls until the analog meter reads "9" (or any other agreed upon value). Adjustment of sensitivity insures that all further readings will be in scale.
D. Set SPECIMEN SWITC~ to BUFFER BLANK.. The digital meter is connected to the output of the variable gain photometer. All other parts of the circuit are disconnected from th~ digital ' meter.
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iSZ4 E. Insert buffer blank t~lbe in optical station and adjust the value oE the digital meter to ~ero using the P~30TOMETER
BLANK subtract controls. For most immunology experiments the coarse PHOTOMETER BL~NI< switch will remain set at MEDIUM.
The error contributed by the buEfer scatter will now be subtracted from all subsequcnt readings, taken on other tubes.
F. Adjust ANTI~ODY BLANK potentiometer fully clockwise to the zero position. This ensures that in the next step the antibody blank subtract will be set at zero.
G. Set SPECIMEN SWITCH to ANTIBODY BLANK, resulting in these occurrences:
i. The integrator is reset to zero and, therefore, input #3 to final amplifier is zero.
ii. Input ~2 to final amplifier is connected to ANTIBODY
BLANK potentiometer which was set at zero.
iii. Digitàl meter is now connected to output of final amplifier. Number displayed is the result of the previous operation, therefore, it is ignored.
H. Insert antibody blank tube in optical station and push COMPUTE. First, the minimum point memory is reset to zero;
then, the minimum point detector will examine the output of the variable gain photometer for a pre-determined period set by t~e COMPUTING TIME control and at the end of that period store the lowest amplitude which has occurred. The digital meter displays that same value, since inputs 2 and
This invention relates to the use of nephelometry to assay the amount of immuno-chemical complex present in a sample.
The invention is based on the fact that in an immuno-chemical complex assay, the various ingredients present have diverse sizes. The particles, including dissolved macromolecules, in buffer, antibody and serum are much smaller than the particles of immuno-chemical complex, while fortuitous particles of dust are generally much larger. As a result of this difference in size and the difference between the large concentration of buffer, antibody and serum present and the small concentration of dust present, it follows that the scatter due to the dust particles fluctuates while the scatter due to all other components is reasonably steady.
Thus in accordance with this invention there is provided in a system for measuring the concentration of medium sized particles in a mixture with both larger and smaller particles, said medium and smaller particles being substantially constant in a macroscopic field of view and said larger particles fluctuating in said field of view, the following combination:
means for directing a beam of electromagnetic radiation through said mixture of particles, means for establishing said field of view, and for sensing and measuring the scatter, in said field of view, from said beam along a direction off-axis to the beam, said scatter being a measure of the combined concentration of all particles present in said mixture, means for suppressing the fluctuatiOn in said measurement, due to the fluctuation of the larger sized particles, by clipping out the fluctuating peaks of the measurement during a period of time, thereby giving a steady ..
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measurement determined by the minimum measurement over said perlod of time, said steady measurement being indicative of the combined concentration o~ medium and small sized particles in said mixture, ;
and means for subtracting from said steady measurement an amount known to represent the concentration of the smaller particles, thereby giving a measurement indicative of the concentration of only said medium sized particles in said mixture.
As a further embodiment of the invention the com-bination described above may also include means for sequentially measuring the concentration of different ones of said smaller ;
sized particles by sequentially sensing scatter from samples of said smaller sized particles, said samples of smaller sized particles being related, in concentration, to the corresponding concentrations in said mixtures of said particles, means sequentially and automatically storing the measurements of concentration of said different smaller sized particles, said storing occurring at the time said sequential measurements are made, and means for utilizing said stored measurements in said means for subtracting.
Further embodiments of the invention comprise a nephelometer and its use for detecting and measuring the quantity of antigens present in a sample as more completely described hereinafter.
The nephelometer itself is characterized by these ;
features:
The optical system uses an arrangement which accepts samples in test tubes and accurately measures forward scatter o~
-a small volume of liquid, spaced within the walls of the test tube.
The design of the Gptical system is such that specular reflection of the incident beam on th~ test tube walls does not interfere with cm/~ ~ 3 ~
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measurement of the scatter, and thus cornmercial test tubes are satisfactory for use as test cells.
The angle of forward scatter is chosen to be about 30 from the illuminatiny beam within said small volume, as it has been found that for a broad range of angles centered on this value, the ratio of desired scatteriny, from the immuno-chemical complex particles of interest to the scattering, not of interest, by the larger and smaller particles that are present in thé assay, is considerably higher than at other scattering angles.
The scatter due to large particles, such as dust, is irregular and is ignored by the electronic circuitry, which measures the minimum value of sca'cter signal over a period of time.
The value of scatter due to particles smaller than those of interest is subtracted from the total scatter to determine the amount of scatter from particles of interest. This subtraction is done semiautomatically by the instrument, in accordance with readings made on the standard or "blank" solutions o the particles ~;
smaller than those o~ interest.
There is described below a certain protocol in ~ ~
accordance with which =he assay 's per/ormed ~
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BRIF,F Dl;SCRi PTION OF VI~;WS OF DR~WING
__ Figure 1 is a vertical cross-sectional view taken through the optical portion of an improved immunoassay nephelo-meter constructed in accordance with the present invention.
Figure 2 is an enlarged horizontal cross-sectional view taken substantially on the line 2-2 of Figure 1.
Figure 3 is an enlarged fragmentary vertical cross- -sectional view of the light-scattering portion of the nephelo- ;~
meter of Figure 1.
Figure 4 is a perspective diagram, corresponding to Figure 3, illustrating how the photomultiplier field of view is restricted by field stops so as to permit it to observe forward scatter from only a limited portion of the sample within the test tube.
Figure 5 is a simplified block diagram of the electrical signal processing and control circuitry of the nephelometer. -Figure 6 is a block diagram of another embodiment of immunochemical complex nephelometry assay system.
Figure 7 is a diagram of the electrical control and measurement sections of the nephelometer, showing the relation-ship of the parts to the front panel controls.
Figure 8 is a perspective view of a preferred em-bodiment of the nephelometer, showing the front panel controls and displays and showing the optical testing station.
Figures 9A and 9B are a schematic of the circuit used in the nephelometer of Figure 8.
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~CKGROUND OF INVENTIOM
_ In the bioloyical laboratory nephelomctry is a standard tool for quantitatively measuring the amount of certain biologicals in a liquid sample. The measurement is made by directing a beam of light through the liquid, and determining the amount of light which is scattered at different angles. The amount which is sca-ttered depends on the size of the scattering particles, their concentration, their shape, the wavelength of the light used, -the refraction indexes of the particles and of the medium in which they are suspended, and the angle at which the scatter is measured. There is a vast body of learning which enables a determination of the concentration to ~e made from such observations, if the dis-tribution of particle sizes is ~nown, but there are practical -difficulties.
For example, in the particular instance which led to the present invention, it was desired to measure the amount of an immunochemical complex in a sample.-From filtration tests made to determine the approxi-mate size of the particles giving rise to the desired and undesired scattering signals, respectively, it was determined that the desired immunochemical complex will pass through a 0.4 micron filter but is stopped by a 0.2 micron filter. Its .. . .
size is therefore, roughly, about 300 nanometers.
The angle chosen for measurement of the scattered -light strongly influences the magnitude of the measurement.
A small forward angle increases the amount of scattered light, as is obvious when considering the appearance of lights at night throllgh a fog. Furthermore, the larger and .
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medium sized particles are relatively more effective in seattering light at small forward anyles than aré the small particles. ~hus, in order to minimize the contribution of small sized particlas, small forward angles are desirable.
~lowever, strong scattering is also obtained from large dust particles which are unavoidably present in the samples. Thus, to minimize the contribution of large sized particles, larger angles of forward scatter should be used.
In making the measurement, the invention seizes on the fact that the particles which are of interest are the largest of the particles present in a constant macroscopic manner. Larger particles, namely, dust, dance around in the so.lution and their scattering is not constant because the dust particles, relatively few in number from a statistical point of view, are not constant in the field of the light beam. .:
Aecordingly, the invention measures the desired eomponent by subtraeting from the observed value the constant eontribution from particles smaller than those of interest while eleetrically suppressing the fluetuating contribution from dust, .
so that it is not included in the measurement.
It has been found that there is a broad range of forward .
seatter angles, eentered at about 30, where the angle chosen inereases the ratio of scatter by medium and large sized partieles to scatter by small sized partieles, without also inereaslng the seatter from the large sized particles to sueh an extent as to o.verload the ability of the electrical system to suppress the ~ fluetuating eomponent representing the large sized partieles.
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D~TAII,ED DESCRIPTION
Referring to the drawings, 11 generally designates the optical p~rtion of a nephelome~er in accordance with the present invention. The nephelometer 11 comprises a housing 12 in which is suitably mounted a horizontally-directed laser unit 13, for exam~le, a Model 155 laser unit, manufactured by Spectro-physics, Mountain View, California, which yenerates a 632.8 nm tnanometer) laser beam, shown at 14.
Housing 12 is provided with a vertical sample-receiving chamber 15 having a relatively thick right side wall 16, as viewed in Figure 1, front and rear walls 17 and 18, a left side wall 19, and a bottom wall 20. Chamber 15 is provided with a removable top cover 21 having a peripheral flange 22.
Right side wall 16 is formed.with an inwardly facing vertical V-groove 23 adapted to be engaged by a conventional vertically positioned test tube 24. A verti.cal pressure block 25 formed with an inwardly facing vertlcal V-groove 26 is :~--slidably positioned in the left side chamber 15, as viewed in .
Figure 1, and is provided with a pair of vertical bowed wire : springs 27,27 secured symmetrically to the left side face of ; the block so as to bear against chamber wall 19 and cause the V-grooved block 25 to exert positioning spring ~orce against the test tube 24 to hold the test tube firmly against V-groove : 23 of wall 16, as shown in Figure 2.
Wall 19 and block 25 are formed with respective laser beam apertures 28 and 29 aligned with beam 14. Wall 16 is formed with a vertically extending recess 30 including a light trap recess 31 aligned with beam 14 and lined with suitable :
light-absorbing material. Recess 30 communicates with a 45- ~ i , , .. .
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inclined li~ht passage 32 formed in wall 16 and provided at its opposite ends ~ith collimating field stops 33 and 3 having 2.8 divergence round field stops as above described aligned at ~5 with respect to beam 14 and aimed, or a conventional 12 x 75 mm test tube 24 at a point on the wall of the test tube about 3.07mm above the beam center line.
Field stop 34 opens into a light tube 35 leading to a photomultiplier tube 36 mounted in a chamber 37 provided therefor on housing 12, as shown in Figure 1.
In some conventional nephelometers utilizing test tubes as test cells, the incident beam and the observed scatter lie in a plane perpendicular to the axis of the test tube.
In such an arrangement, incident rays are multiply reflected from the sides of the test tube in the same plane as the observed scatter and are picked up without discrimination.
One of the features of the instant nephelometer is the placement of the test tube axis in the same plane as that of the incident and scattered rays, thus avoiding the spurious signal due to multiple reflections from the sides of the test tube. These spurious signals are not reproduceable, using different test tubes. The multiple reflections referred to are those at the glass-air interface, and they exist in the absence of imperfectlons, but are ;~
widely different with r~ifferent test tubes because of the large number of reflections involved.
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As can be seen from.Figure 4, the laser beam strongly illuminates a section of the test tube 24 and its contents between entrance 24A and exit 24D. At these two spo-ts, the unavoidable surface deposits and artifacts usually found on and in the glass or plastic of laboratory test tubes or cuvettes and the irregulari-ties of the tube-liquid interface and tube-air interface cause a strong forward scatter, which is unrelated to the forward scatter which it is desired to measure. In order to eliminate this undesired forward scatter from the measurement, field stops.33 and 34 coopera-te to define :~
a family of extreme rays, two of which, 63A and 63B, are -shown. These two extreme rays lie in a vertical plane containing the vertical diameter of each of the field ~.
stops 33 and 34~ These two extreme rays, which are shown as breaking at the rear surface of the test tube, define a part of the front and rear limits of view 24B and 24C.
The portion of the test tube volume between these two ~ ~
limits of view and also illuminated by the laser beam, ;
is accessable to view by the photomultiplier tube 36. :
This tube has an entrance aperture 36B which is larger than, and surrounds the exit aperture 36A of the field -stops 33 and 34.
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l'he field stops _3 and 34 are mounted vertically and therefore define a field of view whose right cross section is elliptical rather than round. This is a matter of convenience .in using round field stops and vertical mountings.
Although the effectively used portion of the tube 24 appears in Fiqure 4 to extend almost from inner tube wall to inner tube wall, the showing is exaygerated to render details visible. In our pre~erred embodiments, we have used an.effective portion about 1 millimeter in major dimension, near the center of a 10 millimeter diameter test tube. This disproportion in size increases the accuracy of measurement.
Designated at 38 (Figure 2) is a safety shutter plate which is slidably engaged against left wall 19 and which has a bottom flange 39 through which is slidably received a headed vertical pin 40 rigidly secured to the bottom wall of housing 12~
A coiled biasing spring 41 surrounds the lower portion of the pin 40 and bears between flange 39 and the housing bottom wall. The .
top end of plate 38 extends through a guide slot 42 in the top wall of housing 12 located beneath the peripheral flange 22 of top cover 21. Shutter plate 38 has a light aperture ~3 which ~ is moved into registration with wall aperture 28 when top cover 21 is seated in closed position ovçr the top end of chamber 15, as shown in Figure i. When the cover 21 is removed, spring 41 ~ :
elevates the shutter plate 38 into blan~ing position covering aperture 28. This insures against dangerous laser flash hazards which may be present when an operator looks downwardly into the test tube 24 with cover 21 removed.
As above e~plained, and as shown in Figures 3 and 4, there is refraction at the wall of test tube 24, so that the .
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exit a~le of the sc~tter beam, sho~l at 63, is 45 to the incid~nt laser be~m 14, whereas inside the test tube the scatter angle is 31.6 to the incident laser beam. I~le angle 45 is a conveni~nt manufacturing angle, .
and the cons~quential angle of 31.6 has been found to give a high ratio of scattering from immuno-chemical complex particles to scattering from smaller particles, without overloading the electrical processing circuit (which will be explain~d belcw) with excessive fluctuating noise signals arrising from the presence of l æge particles, such as dust. It will also be seen that the angled scatter beam seen by the photGmultiplier tube 36 is in a vertical plain containing the axis of the test tube,thereby eliminating artifacts due to internal reflections in the test tube, as absve explained. -Test tubes satisfactory for use ~ith the invention are from commercial stock. For example, ordinary Kimble test tubes, 10 x 75 mm æe satisfactory, unless they have defects in the wall obvious to brlef visual inspection.
In operatiol~, particles of approximately 0.3 micron size in the liquid in test tube 24 scatter the laser beam 14 strongly along the optical path defined by the field stops 33,34 and generate corresponding signals in the photomwltiplier tube 36, so that these signals can be used to measure the a~ount of antigen originally present in the test tube, after a kncwn amount of antibody material is added to the liquid contained in the test tube..................................................... . ?
At low antigen levels and relatively low forward scattering angles, such as the ar~?le employed herein (approximately 31.6) the influence of dust particles in the test tubet and similar artifacts, becomes quite Lmportant and presents.a source of serious error. The drawing, Fi~. 5, illustrates in block fonm a signal-handling circuit which discriminates against sporadic positiv~ spike effects caused by such dust particles or other artifacts. . . . .. ..
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A steady offset voltage greater than the maximum photomultiplier signal is applied at 45 to the other input of inverter/and adder 44B. The input signal at 47 is inverted in the inverter 44A, as shown.at 72, and the inverted peaks are shown at 73. The steady po'sitive input voltage applied at 45 is added in the adder 44B to the signal 72, and the resultant positive output signal at 46, shown at 74, has positive maxima corresponding to the original minimum values 75 of the input signal applied at 47.
The output signal at 46 is'passed through a timer switch element 76, an electronic switch 48, a diode 49 and an operational amplifier 50, providing an output signal at 52 in the form of a positive peak when the variable input at 47 'is a minimum. The greatest positive peak signal during the sampling period reaching operational ampli~ier 50 is held in capac'itor 77 to develop a steady comparison signal, available at 52, which is applied at 78 to one input of a comparator 53. The output s'ignal 74 from device 44 is applied (with timed switch element 76 closed) to the other input 79 of comparator 5~.
.Switch 48 will close initially at the init'ial value of curve 74~ Capacitor 77 will charge to this value, at which point switch 48 will open by the action of comparator 53.
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The value at 52 will be hel~l until ~he curve 7~ amplitude exceeds the initial stored highest point value at 52, at which point switch 48 will again close. The value at 52 will now assymptoti-cally track tincrease) ~he relatively stable positive maximum values of curve 7~, giving a new stored value at 52. Curve 74 will continue to establish such new stored values (with each increase). The comparator ignores the effects of minima caused by artifacts ~suchas Ininima 80). At any time that the amplitude of curve 74 ~oes below the tracked stored value at 52, switch 48 opens, and the signal at 46 is not forwarded to the inte-grating capacitor 77 for storage. Nor can the positive signal stored in capacitor 77 be lost unless reset switch 88 is closed.
These positive signals cannot be lost through diode 49 when the signal at output 46 ls more negative (as at 80) than the value stored in integrating capacitor 77 because of the poling of diode 49, which permits positive charge to flow only from left to right.
The positive signals cannot be lost through operational amplifier 50 because the input impedance of the operational amplifier is enormous, amounting effectively to an open circuit.
Thus, electronic switch 48 opens when the signal at 78 is greater than the signal at 79. The maxima of signal 7~ will, however, be stored in integrating capacitor 77. The stored signal at 52 is thus compared with the instantaneous signal at 46 in the comparator 53, whose output controls switch 48 so that switch 48 is held closed only when the variable input at 47 is higher than a previously stored value. Therefore, switch 48 is normally closed (no signal at 52) and opens when the signaL 74 goes below its prior stored value.
The desired positive antigen scatter signal at 52 is summed in an algebraic summing amplifier 54 with a suitable negative - . .
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offsct signal applied at 8], with a negative antibody blank signal applied at 82, adjusted in a manner presently to be .¦
described, and with a negative stored serum ~antigen) blank .
signal from an integrator 83, applied at 84. The output signal from amplifier 54 i.s delivered through a timed switch element ~5 to a digital display device 56. The disylay provided by device 56 is for a selected period of opera~ion of the algebraic summing amplifier 54, con~rolled by a manually activated computing timer 8fi. Timer 86 is designed to provide a signal sampling period of at least several seconds in order to discriminate adequately against large dust particles or similar artifacts.
When timer 86 is activated by its starting switch, shown at 87, it discharges the signal storage capacitor 77 by momentarily closing a reset switch 88 connected across the ~:
capacitor, closes the timer switch element 76 and opens the ; switch element 85. This provides several seconds for the ac-cumulation of the antigen scatter signal in capacitor 77. At the end of the timed period, switch element 76 opens and closes, producing thè digital display.in device 56.
The negative antibody blank adjustment signal at 82 is obtained from a potentiometer 90 connected to a suitable voltage source. The potentiometer is adjusted ~with its control - switch 91 closed) to provide a negative signal at 82 such as. to give a zero readout on device 56 in a prior test on a blank reference sample of antibody material.
The negative antigen (serum) blank signal at 84 com-prises a compensation signal obtained from the output o~ algebraic summing amplifier 54, with the antibody blank switch 91 open, in a prior test on a plain antigen reference sample before antibody . ' ' ~'' '-' .':', -' . ' ' . .
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material is added~ This ar,tigen tserum) blank signal is stored in integrat:or 83 by activating a "set" switch 93 during such a prior test. Thc stored negative antigen blank signal is then ' available to supply the required compensating negative blank antigen (serum) signal at 84 during the test on the final mix-ture above described.
It will therefore be seen that by subtracting the preset antibody blank signal at 82 and the stored antigen blank signal at 84 from the main test signal at 52, the required discrimination against other particles, such as free antibody and other particles in the antibody blank and free antigen and other particles in the serum blank, is accomplished, and the digital display of the immunocomplex particles is not affected by the presence of said other particles in the final test mixture. This digital display can ther,efore be used to provide an accurate indication of the amount of antigen originally present in a sample after the sample has been exposed to a known quantity of antibody reagent.
The operation of the system of Figure 5 can be under- ,~
stood more easily if it is compared to that of the simpler em-bodiment of Figure 6. These two embodiments differ in that the system of Figure 6 is a four-channel system in which four optical measurements are simultaneously made and simultaneously processed to produce the desired measurement display at 56a. In the system of Figure 5 the corresponding four optical measurements are 'sequentially made, the, results of the first three tests are stored, , and these stored measurements are combined with the fourth measure-ment to give a measurement indication at digital display S6a.
In Figure 6 there are four optical test stations lla to lld. At station llat a forward scatter measurement is made of -.: ' ' ' , ~ , , Z4~
the bufer sol~tion, ~Ihich is ~vailablc in such purity as to contain negligible dust. EaCh of the other biological reagents which must be used, the antibody and the serum, which are measured at stations llb and llc, necessarily have dust in them because of the manner in which they are obtained. At the fourth station there is measured a solution which contains as ingredients both the antibody and the serum sample, which react to produce the immunochemical complex whose concentration is to be indicated at 56a.
The signal at line 101 is one whlch corresponds sub-stantially only to the scattering property of the pure buffer.
Since buffer is in the mixtures at stations llb to lld, and the buffer signal is similarly subtracted from other signals at difference amplifiers 108 to 110, the buffer signal, in effect, ` sets the zero operating point or datum reference or the instrument.
The signals at lines 102 to 104 are irregularly affecte~
by dust, and the dust signals are eliminated by means of the .,; . .
minimum point memory circuits 105 to 107. These circuits correspond to the minimum point memory circuit of Figure 5, comprising inte-grator capacitor 77, diode 49, electronic switch ~8 and comparator 53.
The signals from differential amplifiers 108 to 110, when comb1ned in proper sense in algebraic sum amplifier 54a, produce the immunochemical complex signal which is fed to measurement display . 56a.
It will be seen, by tracing the signals in Figure 6, that each of the undesired signals is eliminated from the output of algebraic sum amplifier 54a, either by cancellation in minimum ; point memories 105 to 107 or by subtraction in differential amplifiers 108 to 110 or algebraic sum amplifier 5~a.
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Fi~urc 7 is a simplified block diagram of the invention showing how the objec~ives ar~ accomplished. No attempt has been made to illustratc ~he actual circuits, but to show Eunctiona~
characteristics. Probably the best way to describe the operation of the system is to follow the protocol and explain what ha~pens as the operator adjusts the knobs or pushes the di~ferent buttons.
For this purpose the diagram shows, adjacent the appropriate place in the circuit, the label which is affixed to the front panel con-trol which is manipulated by the operator during the test.
A significant point that must be remembered for proper understanding is that there are four SPECIMEN SWITCH positions, BUFFER BLANK, ANTIBODY BLANK, SAMPLE BLANK, and SAMPLE READ, which are mutually exclusive.
~ Protocol A. Rfter a 30 minute warm-up, turn HIGH VOLTAGE on. This con~
nects negative voltage to the cathode of the photomultlpliex tube.
B. Lift sample station cover. PHOTOMETER BLANK switch to MEDIUM.
SENSITIVITY switch to X3. Adjust analog meter to zero u~ing the PHOTOMETER BLANK fine controls~ This step establish~s electronic "zero". -C. Put a tube containing the highest "reference" concentration of antigen/antibody into the sample station and adjust the . . .
sensitivity controls until the analog meter reads "9" (or any other agreed upon value). Adjustment of sensitivity insures that all further readings will be in scale.
D. Set SPECIMEN SWITC~ to BUFFER BLANK.. The digital meter is connected to the output of the variable gain photometer. All other parts of the circuit are disconnected from th~ digital ' meter.
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iSZ4 E. Insert buffer blank t~lbe in optical station and adjust the value oE the digital meter to ~ero using the P~30TOMETER
BLANK subtract controls. For most immunology experiments the coarse PHOTOMETER BL~NI< switch will remain set at MEDIUM.
The error contributed by the buEfer scatter will now be subtracted from all subsequcnt readings, taken on other tubes.
F. Adjust ANTI~ODY BLANK potentiometer fully clockwise to the zero position. This ensures that in the next step the antibody blank subtract will be set at zero.
G. Set SPECIMEN SWITCH to ANTIBODY BLANK, resulting in these occurrences:
i. The integrator is reset to zero and, therefore, input #3 to final amplifier is zero.
ii. Input ~2 to final amplifier is connected to ANTIBODY
BLANK potentiometer which was set at zero.
iii. Digitàl meter is now connected to output of final amplifier. Number displayed is the result of the previous operation, therefore, it is ignored.
H. Insert antibody blank tube in optical station and push COMPUTE. First, the minimum point memory is reset to zero;
then, the minimum point detector will examine the output of the variable gain photometer for a pre-determined period set by t~e COMPUTING TIME control and at the end of that period store the lowest amplitude which has occurred. The digital meter displays that same value, since inputs 2 and
3 of final amplifier are zero.
I. Adjust ANTIBODY BLANK potentiometer CCW until the digital meter reads zero. Voltage at pin 2 of final amplifier is ~ . increased until it equals the value at~the output of the minimum point detector . Since that value is the valuc of _ /f_ ' . .
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the antibody blank, the antibody blank value is permanentlyrecorded in the potentiometer.
J. Set S~ECIMEN switch to S~MPLE ~ANK. The digital meter still reads the output of the final amplifier, the integrator is stili set to zero and the ANTIBODY BLANK potentiometer is disabled. The number displayed is the value of the antibody blank obtained in previous steps.
K. Insert the sample blank tube into the optical station and push COMPUTE. The minimum point memory is reset to zero. The minimum point detector examines the output of the variable gain photometer and stores the minimum point. The number displayed in the digital meter is, therefore, the value of the serum blank.
L. SPECIMEN switch is set to SAMPLE READ. ~he digital meter still reads the output of the final amplifler. The action of going to "read" triggers the 0.5 second ti~er which - controls contacts Kla and Klb. Contact Kla shorts input #2 of final amplifier for the first 0.5 sec. Contact Klb connects , the input of the 1ntegrator to the output of the final amplifier; as a result, input #3 of the final amplifier will gradually increase until it is equal to input #1. (Remember input #2 is still zero.) The digital meter will, therefore, go to a reading of 000Ø At the end of the 0.5 second period, Klb and KIa will open. The value at input #3 will be held and ~
will correspond to the antibody blank value since the antibody ~ ;
blank potentiometer is also connected. Since input #l is also the serum blank value, the meter now displays a negative antibody blank value.
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~1. Insert tube containin~ the antigen/antibody immunochemical complex in the read station and press COMPUTE. The minimum point detector is first reset to zero. Then it examines the value of the variable gain photometer and stores the ~inimum point which is fed to i.nput ~1 of final amplifier. This value corresponds to the antigen/antibody immunochemical complex concentration plus the serum and the antibody blank.
Since pins 2 and 3 contain the previously obtained values of antibody and serum blanks, respectively, the value obtained at the meter is the immunochemical complex concentration.
N. Record reading of METER. This is the only number the technician records.
A prototype or preferred embodiment of the invention has recently been built. This is shown in Figures a, 9A and 9B.
Figure 8 shows, in perspective, the external view of the instrument. On the left side is the electrical console 101 and on the right is the optical unit lle. Thè latter has a cover 21a which is hinged at the rear edge, unlike the flanged cover 21 of the embodiment of Figure 1. A hinged cover is obviously more convenient, as it can merely be swung up,back and out of the way on its hinges, as shown by the two dotted lines o~
Figure 8, and does not have to be manually removed. The photo-multiplier unit, with its high voltage connector projecting tothe rear, is at 37a.
The console 101 has a control panel 102 with a number of controls. These controls are labeled, in the Figure, with their designations, so as to correspond with the designations in Figure 7, and with the designations in the Protocol which was given previously.
Figures 9A and 9B show the wi.ing diagram in greater . . .
detail than in Figure 7. It will be noted tha~ each of the four-, . ~ . ' .
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SPECIMEN SELECT push buttons and the DAMPING push buttons arP
self lighting when actuated, thus assisting the operator to keep track of his operations. ~11 of the electronic circuitry is strai~htforward and uses common components and its action will be self-evident in the light of the previous explanations to the person skilled in the art.
While specific embodiments of an improved rnethod and apparatus have been disclosed in the ~oregoing descrip~iorl, it will be understood that vaxious modifica-tions within the spirit of the invention may occur to those skilled in the art.
Therefore it is intended that no limitations be placed on the invention except as defined by the scope of the appended claims.
This application is a division of copending Canadian , application Serial No. 239,752 filed November 17, 1975 and is , .
related to a companion application Serial No. 239,784 filed November 17, 1975 for "Nephelometer having means Semiautomatically ~ ~ `
, Cancelling Components from Scattering by Particles Smaller or Larger than those of Interest" (Rl~dolfo R. Rodriq~lez).
~:
.
, : - - - .: . .
, ~ , , , :
- ,
I. Adjust ANTIBODY BLANK potentiometer CCW until the digital meter reads zero. Voltage at pin 2 of final amplifier is ~ . increased until it equals the value at~the output of the minimum point detector . Since that value is the valuc of _ /f_ ' . .
.. ,., . ~ , : : .
-: ' , , , : ' ' ' ' : ~. ~
' . , . ' ':, , , . '' .
the antibody blank, the antibody blank value is permanentlyrecorded in the potentiometer.
J. Set S~ECIMEN switch to S~MPLE ~ANK. The digital meter still reads the output of the final amplifier, the integrator is stili set to zero and the ANTIBODY BLANK potentiometer is disabled. The number displayed is the value of the antibody blank obtained in previous steps.
K. Insert the sample blank tube into the optical station and push COMPUTE. The minimum point memory is reset to zero. The minimum point detector examines the output of the variable gain photometer and stores the minimum point. The number displayed in the digital meter is, therefore, the value of the serum blank.
L. SPECIMEN switch is set to SAMPLE READ. ~he digital meter still reads the output of the final amplifler. The action of going to "read" triggers the 0.5 second ti~er which - controls contacts Kla and Klb. Contact Kla shorts input #2 of final amplifier for the first 0.5 sec. Contact Klb connects , the input of the 1ntegrator to the output of the final amplifier; as a result, input #3 of the final amplifier will gradually increase until it is equal to input #1. (Remember input #2 is still zero.) The digital meter will, therefore, go to a reading of 000Ø At the end of the 0.5 second period, Klb and KIa will open. The value at input #3 will be held and ~
will correspond to the antibody blank value since the antibody ~ ;
blank potentiometer is also connected. Since input #l is also the serum blank value, the meter now displays a negative antibody blank value.
. ' ' ' ' .
:., ~ , . - ' ' : - ., , :-............... . ,, ,:,. . , :
; , ' : , . ' ' ' ' ' ' :
~1. Insert tube containin~ the antigen/antibody immunochemical complex in the read station and press COMPUTE. The minimum point detector is first reset to zero. Then it examines the value of the variable gain photometer and stores the ~inimum point which is fed to i.nput ~1 of final amplifier. This value corresponds to the antigen/antibody immunochemical complex concentration plus the serum and the antibody blank.
Since pins 2 and 3 contain the previously obtained values of antibody and serum blanks, respectively, the value obtained at the meter is the immunochemical complex concentration.
N. Record reading of METER. This is the only number the technician records.
A prototype or preferred embodiment of the invention has recently been built. This is shown in Figures a, 9A and 9B.
Figure 8 shows, in perspective, the external view of the instrument. On the left side is the electrical console 101 and on the right is the optical unit lle. Thè latter has a cover 21a which is hinged at the rear edge, unlike the flanged cover 21 of the embodiment of Figure 1. A hinged cover is obviously more convenient, as it can merely be swung up,back and out of the way on its hinges, as shown by the two dotted lines o~
Figure 8, and does not have to be manually removed. The photo-multiplier unit, with its high voltage connector projecting tothe rear, is at 37a.
The console 101 has a control panel 102 with a number of controls. These controls are labeled, in the Figure, with their designations, so as to correspond with the designations in Figure 7, and with the designations in the Protocol which was given previously.
Figures 9A and 9B show the wi.ing diagram in greater . . .
detail than in Figure 7. It will be noted tha~ each of the four-, . ~ . ' .
.. ... .
- : : ' . ':
~L~8~5Z~
SPECIMEN SELECT push buttons and the DAMPING push buttons arP
self lighting when actuated, thus assisting the operator to keep track of his operations. ~11 of the electronic circuitry is strai~htforward and uses common components and its action will be self-evident in the light of the previous explanations to the person skilled in the art.
While specific embodiments of an improved rnethod and apparatus have been disclosed in the ~oregoing descrip~iorl, it will be understood that vaxious modifica-tions within the spirit of the invention may occur to those skilled in the art.
Therefore it is intended that no limitations be placed on the invention except as defined by the scope of the appended claims.
This application is a division of copending Canadian , application Serial No. 239,752 filed November 17, 1975 and is , .
related to a companion application Serial No. 239,784 filed November 17, 1975 for "Nephelometer having means Semiautomatically ~ ~ `
, Cancelling Components from Scattering by Particles Smaller or Larger than those of Interest" (Rl~dolfo R. Rodriq~lez).
~:
.
, : - - - .: . .
, ~ , , , :
- ,
Claims (12)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. In a system for measuring the concentration of medium sized particles in a mixture with both larger and smaller particles, said medium and smaller sized particles being sub-stantially constant in a macroscopic field of view and said larger particles fluctuating in said field of view, means for directing a beam of electromagnetic radiation through said mixture of particles, means for establishing said field of view, and for sensing and measuring the scatter, in said field of view, from said beam along a direction off-axis to the beam, said scatter being a measure of the combined concentration of all particles present in said mixture, means for suppressing the fluctuation in said measure-ment, due to the fluctuation of the larger sized particles, by clipping out the fluctuating peaks of the measurement during a period of time, thereby giving a steady measurement determined by the minimum measurement over said period of time, said steady measurement being indicative of the combined concentration of medium and small sized particles in said mixture, and means for subtracting from said steady measurement an amount known to represent the concentration of the smaller particles, thereby giving a measurement indicative of the concentration of only said medium sized particles in said mixture.
2. The system of claim 1 in combination with means for sequentially measuring the concentration of different ones of said smaller sized particles by sequentially sensing scatter from samples of said smaller sized particles, said samples of smaller sized particles being related, in concentration, to the corresponding concentrations in said mixtures of said particles, means sequentially and automatically storing the measurements of concentration of said different smaller sized particles, said storing occurring at the time said sequential measurements are made, and means for utilizing said stored measurements in said means for subtracting.
3. The system of claim 1 wherein the means for directing a beam of electromagnetic radiation is a laser, the mixture is a liquid contained in a test tube, in combination with an open ended chamber having resilient positioning means to position said test tube in the beam of the laser, said chamber having an entrance aperture in light tight relationship with said laser and having an exit aperture in light tight relationship with a light trap, said entrance and exit apertures being aligned with the laser beam to respectively receive the un-deflected beam before and after it impinges on the test tube, said chamber also having a sensing aperture in light tight relationship with said means for sensing and measuring the said off-axis scatter from said beam, said chamber having a moveable shutter between the laser and the entrance aperture to occult the laser beam, said chamber having a light tight cover for its open end, and means to automatically close said shutter whenever said cover is open and to open said shutter only when said cover is fully closed.
4. The system of claim 3 wherein the said means for establishing said field of view and for sensing and measuring said scatter includes a photodetector in a light tight housing, two field limiting stops on the optical axis of the said scatter, between said test tube and said photodetector, said field limiting stops limiting the sensing of scattered light by said photodetector to a portion of the total light which is scattered in the test tube, said portion coming from the volume within the test tube which is defined by the limits of the laser beam and by the limits defined by the field stops, whereby dirt on and imperfections in the walls of the test tube or proximity effects of the inner wall of the test tube on the particles do not substantially affect the photodetector response.
5. The system of claim 2 wherein said means for sequentially determining and said means for sequentially and automatically storing the scatter from different ones of said smaller sized particles includes means for exposing a standardized sample of one kind of smaller sized particles to the field of view, and means for subtracting from the output of the smaller measurement means a settable amount sufficient to reduce the reading of said scatter measurement to zero.
6. The system of claim 5 in which said standardized sample is sufficiently pure as to be substantially dust free.
7. The system of claim 5 in which said standardized sample contains dust.
8. The system of claim 5 in which said standardized sample is an unknown, collected under known conditions, which is to be assayed.
9. The system of claim 5 in which the means to sub-tract comprises an adjustable-voltage source.
10. The system of claim 2 wherein said means for sequentially determining and said means for sequentially and automatically storing the scatter from different ones of said smaller sized particles includes means for exposing a standardized sample of one kind of smaller sized particles to the field of view, and means for storing the output of the scatter measure-ment means in an electrical integrator.
11. The system of claim 1 wherein the said mixture is contained in a test tube and said means for directing a beam of electromagnetic radiation through said mixture of particles further includes:
means for positioning said test tube within the said field of view;
the longitudinal axis of the test tube, when so positioned, and the longitudinal axis of said beam of electro-magnetic radiation defining a plane and wherein said off-axis scatter is measured along an axis which also lies in said plane.
means for positioning said test tube within the said field of view;
the longitudinal axis of the test tube, when so positioned, and the longitudinal axis of said beam of electro-magnetic radiation defining a plane and wherein said off-axis scatter is measured along an axis which also lies in said plane.
12. The system of claim 1 in which the said medium sized particles of interest are immuno-chemical complex particles, and in which said scatter is forward scatter, and is measured along an axis which lies approximately 30° from the axis of the beam.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA342,031A CA1086524A (en) | 1975-01-29 | 1979-12-17 | Nephelometer having means semiautomatically cancelling components from scattering by particles smaller or larger than those of interest |
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US54506675A | 1975-01-29 | 1975-01-29 | |
| US545,066 | 1975-01-29 | ||
| US60078775A | 1975-07-31 | 1975-07-31 | |
| US600,787 | 1975-07-31 | ||
| CA342,031A CA1086524A (en) | 1975-01-29 | 1979-12-17 | Nephelometer having means semiautomatically cancelling components from scattering by particles smaller or larger than those of interest |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1086524A true CA1086524A (en) | 1980-09-30 |
Family
ID=27166516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA342,031A Expired CA1086524A (en) | 1975-01-29 | 1979-12-17 | Nephelometer having means semiautomatically cancelling components from scattering by particles smaller or larger than those of interest |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1086524A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116793907A (en) * | 2022-03-16 | 2023-09-22 | 上海勘测设计研究院有限公司 | Multidirectional diffraction scattering type particle size analyzer and particle detection method |
-
1979
- 1979-12-17 CA CA342,031A patent/CA1086524A/en not_active Expired
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116793907A (en) * | 2022-03-16 | 2023-09-22 | 上海勘测设计研究院有限公司 | Multidirectional diffraction scattering type particle size analyzer and particle detection method |
| CN116793907B (en) * | 2022-03-16 | 2024-05-14 | 上海勘测设计研究院有限公司 | Multidirectional diffraction scattering type particle size analyzer and particle detection method |
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