CA1076386A - 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
- Publication number
- CA1076386A CA1076386A CA239,752A CA239752A CA1076386A CA 1076386 A CA1076386 A CA 1076386A CA 239752 A CA239752 A CA 239752A CA 1076386 A CA1076386 A CA 1076386A
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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 immuno-chemical 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 immuno-chemical 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
`~ 107638~
RIEF SUMMARY OF INV~NTION
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, an-tibody 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 dif~erence 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 whiie the scatter due to all other components is reasonably steady.
Thus in accordance with this inyention 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 ie1d 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 ~articles, 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 fluctuatin in said measurement, due to the fluc-tuation of the larger sized particles, by clipping out the fluctu~ting peaks of the measurement during a period of time, thereby giving a steady ~.'~ cm/?~ - 2 - ~
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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, thexeby 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 partlcles 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.
~0 Further embodiments of the invention comprise a nephelometer and i`ts use for detecting and measuring the quantity of antigens present in a sample as more completely described herèinafter.
~ 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 of a small volume of liquid, spaced within the walls of the test tube.
The design of the optical sys-tem is such that specular reflection of the lncident beam on the test tube walls does not interEere with -;~ cm/_^ - 3 -. :
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1~76386 measurement of the scatter, and thus commercial test tubes are s~tisfactory for use as test cells.
The angle of forward-scatter is chosen to be about 30 from the illuminating 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 scattering, from the immuno-chemical complex particles of interest to the scattering, not of interest, by the larger and smaller particles that are present in the assay, is considerably higher than at other scattering angles.
~ he scatter due to large particles, such as dust, is irregular and is ignored by the electronic circuitry, which measures the minimum value of scatter 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 of the particles smaller than those of interest.
There is described below a certain protocol in accordance with which the assay is performed.
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7638~i BRIEF DESCRI.PTION OF ~IIEWS OF DRI~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 vie~ 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 simplif~ed 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 diaaram of the electrical control and measurement sections of the nephelometer, sho~ing 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 9~ are a schematic of the circuit used in the nephelometer of Figure 8.
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BACKGROUND OF INVENTION
In the biological laboratory nephelometry 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 scattered 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 be made from sucn observations, if the dis-tribution of particle sizes is known, but there are practical difficulties.
For example, in the par~icular instance which led to the present invention, it was desired to measure the àmount of an immunochemical complex in a sample.
From filtration tests made to determine the approxi-mate size of the particles giving rise to ~he desired and undèsired scattering signals, respectively, it was determined that the desired i~munochemical complex will pass through a 0.4 micron filter but is stopped by a 0.2 micron filtex. 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 li~hts at night through a fog. Furthermore, the larger and .
,, medium sized particles are relatively more effective in scattering light at small forward angles than are the small particles. Thus, in order to minimize the contribution of small sized particlas, small forward angles are desirable.
However, strong scattering is also obtained from large dust particles which are unavoidably present in the samples. Thus, to minimize the contribu~ion of large sized particles, larger angles of forward scatter should be used.
- In making the measurement, the invention selzes 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 solution 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.
Accordingly, the invention measures the desired component by subtracting from the observed value the constant contribution from particles smaller than those of interest while electrically suppressing the fluctuating contribution from dust, so that it is not included in the measurement.
It has been found that there is a broad range of forward scatter angles, centered at about 30, where the angle chosen increases the ratio of scatter by medium and large sized particles to scatter by small sized particles, without also increasing the scatter from the large sized particles to such an extent as to overload the abiiity of the electrical system to suppress the fluctuating component representing the large sized particles.
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~(~763~6 _~TAILED DESCRIPTION
. ~eferring to the drawings, 11 generally designates the optical portion of a nephelometer 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 example, a Model 155 laser unit, manufactured by Spectro-physics, Mountain View, California, which generates a 632.8 nm ~nanometer) laser beam, shown at 14.
` ~ousing 12 is provided with a vertical sample-receivingchamber lS 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 vertical pressure block 25 formed with an inwardly facing vertical 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 sïde face of the block so as to bear against chamber wall 19 and cause the V-grooved block 25 to exert positioning spring force against .. the test tube 24 to hold the test tube firmly against V-groove 23 of wall 16, as shown in Figure 2.
Wali 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 wi~h suitable . light-absorbing material. ~ecess 30 communicates with a 45-. .
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10763t36 inclined light passàge 32 formed in wall 16 and provided at its opposite ends with collimating field stops 33 and 34 having 2.8 divergence round field stops as above described aligned at 45 with respect to beam 14 and aimed, for 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.
Yield stop 34 opens into a liyht 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 imperfections, but are widely different with different test tubes because of the large number of reflections involved.
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 SpotS, 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 cooperate 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 illùminated by the laser beam, is accessable to view by the photomultiplier tube 36.
~his tube has an entrance aperture 36B which is larger than, and surrounds the exit aperture 36A of the field stops 33 and 3~.
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107631~6 The field stops 33 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 Figure 4 to extend almost from inner tube wall to inner tube wall, the showing is exaggerated to render details visible. In our preferred 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 loca~ed beneath the peripheral flange 22 of top cover 21. Shutter plate 38 has a light aperture 43 which is moved into registration with wall aperture 28 when top cover 21 is seated in closed position over the top end of chamber 15, as shown in Figure 1. When the cover 21 is removed, spring 41 èlevates 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 explained, and as shown in Figures 3 and 4, there is refraction at the wall of test tube 24, so that the ' . ' ' :''' : ' .:, ,,, : . ., ': ' ; ' . ' ~C~7~3136 exit angle of the scatter beam, shcwn at 63, is 45 to the incident laser beam 14, whereas inside the test tube the scatter angle is 31.6 to the incident laser beam. The angle 45 is a convenient manufacturin9 angle, and the consequential angle of 31.6 has been found to give a high ratio of scattering frcm immuno-chemical canplex particles to scattering from smaller particles, without overloading the electrical processing circuit (which will be explained below) with excessive fluctuating noise signals arrising fram the presence of large particles, such as dust. It will also be seen that the angled scatter beam seen by the photomultiplier 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 above explained.
Test tubes satisfactory for use with the invention are from commercial stock. For example, ordinary K~ble test tubes, 10 x 75 ~m, are satisfactory, unless they have defects in the wall obvious to brief ~risual inspection.
In operation, 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 photom~ltiplier tube 36, so that these signals can be used to measure the amount of antigen originally present in the test tube, after a known amount of antibcdy material is added to the liquid contained in the test tube.
A~ low antigen levels and relatively low forward scattering angles, such as the angle employed herein (approximately 31.6) the influence o dust particles in the test tube, and similar artifacts, becomes quite important and presents a source of serious error. The drawing, Fig. 5, illustrates in block fonm a signal-handling circuit which discri~inates against sporadic positive spike effects caused by such dust particles or other artifacts.
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. .:, :: , ,: , ,, :, This circuit is a signal prccessing circuit which clips out positive peaks in the photamultiplier output signal, so that said positive peaks never get sent on to and processed by the measuring equipment. These positive peaks are generated by the dust particles in the liquid under examination, or the other above-mentioned artifacts. Thus, because of dust particles, for example, the photometer signal generated in photomultiplier tube 36 is variable, with large positive spikes 71 caused by the individual dust particles. The variable photomultiplier signal is applied at 47 to one input of an inverter/and adder 44s.
` ~ steady offset voltage greater than the maximum photomultiplier signal is applied at 45 to the other input of inverter/and adder 44~- 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 positive 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 amplifier 50 is held in capacitor 77 to develop a steady comparison signal, available at 52, which is applied at 78 to one input of a comparator 53. The output signal 74 from device 44 is applied (with timed switch element 76 closed) to the other input 79 of comparator 53.
Switch 48 will close initially at the initial 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 the eurve 74 amplitude exceeds the initial stored high~st point value at 52, at whieh point switch 48 will again close. The value at 52 will now assymptoti-eally track (increase) the relatively stable positive maximum values of curve 74, 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 minima 80). At any time that the amplitude of eurve 74 goes 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 is 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 cireuit.
Thus, electronic switch 48 opens when the signal at 78 is greater than the signal at 79. The maxima of signal 74 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 7 goes below its prior stored value.
The desired positive antigen scatter signal at 52 is summed in an algebraic summinc; amplifier 54 with a suitable negative .
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'107~;3ll6 offset 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 is delivered through a timed switch element 85 to a digital display device S6. The display provided by device 56 is for a selected period of operation of the algebraic sum~ing ampiifier 54, controlled 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. ~t the end of the timed period, switch element 76 opens and closes, producing the digital display in device 56.
The negative antibody blank adjust~ent 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 prlor 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 of algebraic summing amplifier 54, with the antibody blank switch 91 open, in a prior test on a plàin antigen reference sample before antibody - . , ,. , , : :. , , , . ~ , , : . , :
~0,63~6 material is added. This antigen (serum) blank signal is stored in integrator 83 by activating a "set" switch 93 during such a prior test. The 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 abo~e 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 therefore be used to provide an accurate indication of the amount of antigen origïnally present in a sample after the sample has been exposed to a known quantity of antihody 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 56a.
` ` In Figure 6 there are four optical test stations lla to lld. At station lla, a forward scatter measurement is made of ~: 1 : , ,, .. . .:, . .
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the buffer solution, which is available 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 con~ains as ingredients both the antibody and the serum sample, which react to produce the im~unochemicai complex whose concentration is to be indicated at 56a.
The signal at line 101 is one which 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 for the instrument.
The signals at lines 102 to 104 are irregularly affected by dust, and the dust signals are el-iminated by means of the minimum point memory circuits 105 to 107. These circuits correspond to the minimum point memory circuit o Figure 5, comprising inte-grator capacitor 77, diode 49, electronic switch 48 and comparator 53.
The signals from differential amplifiers 108 to 110, when combined 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 54a.
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Figure 7 is a simplified block diagram Oe the invention showing how the objectives are accomplished. No attempt has been made to illustrate the actual circuits, but to sho~ functional characteristics. Proba~ly the best way to describe the operation oE the system is to follow the protocol and explain ~hat ha~pens as the operator adjusts the knobs or puslles the difEcrent buttons.
Yor this purpose the diagram shows, adjacent the approprlate place in th~ circuit, the Label which is af~i~ed to the front panel con-trol which is manipulated by the operator during the test.
A significant point that must ~e remembered for proper understanding is that there are four SPECII~IEN SWITC~I positions, BUFFER BL~NK, I~NTIE30DY BLAN~, S~lPLE BL~NK, and S~IPLE E~EAD, which are mutually e~clusive.
Pro~ocol A. ~fter a 8n minute warm-up, turn E~IGH ~T0LTAGE on. This con-nects negative voltage to the cathode of the photomultiplier tube.
B. Lift sc~mple station cover. PHOTOMETER BLANK switch to MEDIU~I.
~ SE~SITIVITY switch to X3. Adjust analog n:eter to zero using ; the PEIOTO~TER BLANK fine controls. This step establish~s electronlc "zero".
C. Put a tube containing the highest "reference" concentration of antigen/antibody into the sample station and adjust the s~nsitivity controls until the analog meter reads "9" (or any other agreed upol; value). Adjustment of sensitivity insures that all further readings will be in scale.
D. Set SPECI;~lEN SWITCEI to BUE~FER 8L~NK. The digital meter is connected to the output of the variable gain photometer. All other parts o~ tlle circuit are disconnected from the d~ital meter.
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~76386 Insert buffer blank t~lbe in optical station and adjust the value of the digital meter to zero using the PHOTOMETER
BLANK subtract controls. For most immunology experiments the coarse PHOTOMETER BLANK switch will remain set at MEDIUM.
The error contributed by the buffer scatter will now be subtracted from all subsequent readings, taken on other tubes.
F. Adjust ANTIBODY 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 ANTIsoDY
~LANK potentiometer which was set at zero.
iii. Digital 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 biank tube in optical station and pu;h 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 the 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 ~NTIBODY BLANK potentiometer CCW until the digital meter reàds zero. Voltage at pin 2 of final amplifier is increased until it equals the value at the output of the minimum point detector . Since tllat value is the value of : . , : . . , :, 1~176386 the antibody blank, the antibody blank value is permanently recorded in the potentiometer.
J. Set SPECIMEN switch to SA~PLE BLANK. The di~ital meter still reads the output of the final amplifier, the inte~rator is stili set to zero and the ANTIBODY BLANK potentiometer is disabled. The number dlsplayed is the value of the antibody blank obtained in previous steps.
X. Insert the sample blank tube into the optical station and push COMPUTE. The minimum point memory is reset to æero. 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.
. SPECIMEN switch is set to SP~E READ. The digital meter still reads the output of the final amplifier. The action of going to "read" triggers the 0.5 second timer wllich controls contacts Kla and Klb. Contact Xla shorts input #2 of final amplifier for the first 0.5 sec. Contact Klb connects the input of the integrator to the output of the final amplifier; as a result, in?ut #3 of the final amplifier will gradually increase until it is equal to input ~ 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 Rla 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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lU76386 ~1. Insert tube containing 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 minimum point which is fed to input #l 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 ~ETER. Thls is the only nùmber the technician secords.
A prototype or preferred embodiment of the invention has recently been built. This is shown in Figures 8, 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. The latter has a cover 21a which is hinged at the rear edge, unlike the flanged cover 21 Qf 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 oE
Figure 8, and does not have to be manually removed. The photo-multiplier unit, with its high voltage connector projecting to the 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 wiring diagram in ~reater detail than in Figure 7. It will be noted that each of the four SPECIMEN SELECT push buttons and the DAMPING push buttons are self lighting when actuated, thus assisting the operator to keep track of his operations. All of the electronic circuitry is straightforward 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 method and apparatus have been disclosed in the foregoing descrip~ion, it will be understood that various modifications 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 oE the appended claims.
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RIEF SUMMARY OF INV~NTION
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, an-tibody 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 dif~erence 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 whiie the scatter due to all other components is reasonably steady.
Thus in accordance with this inyention 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 ie1d 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 ~articles, 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 fluctuatin in said measurement, due to the fluc-tuation of the larger sized particles, by clipping out the fluctu~ting peaks of the measurement during a period of time, thereby giving a steady ~.'~ cm/?~ - 2 - ~
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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, thexeby 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 partlcles 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.
~0 Further embodiments of the invention comprise a nephelometer and i`ts use for detecting and measuring the quantity of antigens present in a sample as more completely described herèinafter.
~ 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 of a small volume of liquid, spaced within the walls of the test tube.
The design of the optical sys-tem is such that specular reflection of the lncident beam on the test tube walls does not interEere with -;~ cm/_^ - 3 -. :
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1~76386 measurement of the scatter, and thus commercial test tubes are s~tisfactory for use as test cells.
The angle of forward-scatter is chosen to be about 30 from the illuminating 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 scattering, from the immuno-chemical complex particles of interest to the scattering, not of interest, by the larger and smaller particles that are present in the assay, is considerably higher than at other scattering angles.
~ he scatter due to large particles, such as dust, is irregular and is ignored by the electronic circuitry, which measures the minimum value of scatter 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 of the particles smaller than those of interest.
There is described below a certain protocol in accordance with which the assay is performed.
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7638~i BRIEF DESCRI.PTION OF ~IIEWS OF DRI~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 vie~ 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 simplif~ed 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 diaaram of the electrical control and measurement sections of the nephelometer, sho~ing 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 9~ are a schematic of the circuit used in the nephelometer of Figure 8.
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BACKGROUND OF INVENTION
In the biological laboratory nephelometry 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 scattered 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 be made from sucn observations, if the dis-tribution of particle sizes is known, but there are practical difficulties.
For example, in the par~icular instance which led to the present invention, it was desired to measure the àmount of an immunochemical complex in a sample.
From filtration tests made to determine the approxi-mate size of the particles giving rise to ~he desired and undèsired scattering signals, respectively, it was determined that the desired i~munochemical complex will pass through a 0.4 micron filter but is stopped by a 0.2 micron filtex. 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 li~hts at night through a fog. Furthermore, the larger and .
,, medium sized particles are relatively more effective in scattering light at small forward angles than are the small particles. Thus, in order to minimize the contribution of small sized particlas, small forward angles are desirable.
However, strong scattering is also obtained from large dust particles which are unavoidably present in the samples. Thus, to minimize the contribu~ion of large sized particles, larger angles of forward scatter should be used.
- In making the measurement, the invention selzes 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 solution 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.
Accordingly, the invention measures the desired component by subtracting from the observed value the constant contribution from particles smaller than those of interest while electrically suppressing the fluctuating contribution from dust, so that it is not included in the measurement.
It has been found that there is a broad range of forward scatter angles, centered at about 30, where the angle chosen increases the ratio of scatter by medium and large sized particles to scatter by small sized particles, without also increasing the scatter from the large sized particles to such an extent as to overload the abiiity of the electrical system to suppress the fluctuating component representing the large sized particles.
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~(~763~6 _~TAILED DESCRIPTION
. ~eferring to the drawings, 11 generally designates the optical portion of a nephelometer 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 example, a Model 155 laser unit, manufactured by Spectro-physics, Mountain View, California, which generates a 632.8 nm ~nanometer) laser beam, shown at 14.
` ~ousing 12 is provided with a vertical sample-receivingchamber lS 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 vertical pressure block 25 formed with an inwardly facing vertical 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 sïde face of the block so as to bear against chamber wall 19 and cause the V-grooved block 25 to exert positioning spring force against .. the test tube 24 to hold the test tube firmly against V-groove 23 of wall 16, as shown in Figure 2.
Wali 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 wi~h suitable . light-absorbing material. ~ecess 30 communicates with a 45-. .
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10763t36 inclined light passàge 32 formed in wall 16 and provided at its opposite ends with collimating field stops 33 and 34 having 2.8 divergence round field stops as above described aligned at 45 with respect to beam 14 and aimed, for 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.
Yield stop 34 opens into a liyht 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 imperfections, but are widely different with different test tubes because of the large number of reflections involved.
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 SpotS, 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 cooperate 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 illùminated by the laser beam, is accessable to view by the photomultiplier tube 36.
~his tube has an entrance aperture 36B which is larger than, and surrounds the exit aperture 36A of the field stops 33 and 3~.
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107631~6 The field stops 33 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 Figure 4 to extend almost from inner tube wall to inner tube wall, the showing is exaggerated to render details visible. In our preferred 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 loca~ed beneath the peripheral flange 22 of top cover 21. Shutter plate 38 has a light aperture 43 which is moved into registration with wall aperture 28 when top cover 21 is seated in closed position over the top end of chamber 15, as shown in Figure 1. When the cover 21 is removed, spring 41 èlevates 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 explained, and as shown in Figures 3 and 4, there is refraction at the wall of test tube 24, so that the ' . ' ' :''' : ' .:, ,,, : . ., ': ' ; ' . ' ~C~7~3136 exit angle of the scatter beam, shcwn at 63, is 45 to the incident laser beam 14, whereas inside the test tube the scatter angle is 31.6 to the incident laser beam. The angle 45 is a convenient manufacturin9 angle, and the consequential angle of 31.6 has been found to give a high ratio of scattering frcm immuno-chemical canplex particles to scattering from smaller particles, without overloading the electrical processing circuit (which will be explained below) with excessive fluctuating noise signals arrising fram the presence of large particles, such as dust. It will also be seen that the angled scatter beam seen by the photomultiplier 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 above explained.
Test tubes satisfactory for use with the invention are from commercial stock. For example, ordinary K~ble test tubes, 10 x 75 ~m, are satisfactory, unless they have defects in the wall obvious to brief ~risual inspection.
In operation, 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 photom~ltiplier tube 36, so that these signals can be used to measure the amount of antigen originally present in the test tube, after a known amount of antibcdy material is added to the liquid contained in the test tube.
A~ low antigen levels and relatively low forward scattering angles, such as the angle employed herein (approximately 31.6) the influence o dust particles in the test tube, and similar artifacts, becomes quite important and presents a source of serious error. The drawing, Fig. 5, illustrates in block fonm a signal-handling circuit which discri~inates against sporadic positive spike effects caused by such dust particles or other artifacts.
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. .:, :: , ,: , ,, :, This circuit is a signal prccessing circuit which clips out positive peaks in the photamultiplier output signal, so that said positive peaks never get sent on to and processed by the measuring equipment. These positive peaks are generated by the dust particles in the liquid under examination, or the other above-mentioned artifacts. Thus, because of dust particles, for example, the photometer signal generated in photomultiplier tube 36 is variable, with large positive spikes 71 caused by the individual dust particles. The variable photomultiplier signal is applied at 47 to one input of an inverter/and adder 44s.
` ~ steady offset voltage greater than the maximum photomultiplier signal is applied at 45 to the other input of inverter/and adder 44~- 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 positive 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 amplifier 50 is held in capacitor 77 to develop a steady comparison signal, available at 52, which is applied at 78 to one input of a comparator 53. The output signal 74 from device 44 is applied (with timed switch element 76 closed) to the other input 79 of comparator 53.
Switch 48 will close initially at the initial 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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1~)763Y~ I
The value at 52 will be hel~l until the eurve 74 amplitude exceeds the initial stored high~st point value at 52, at whieh point switch 48 will again close. The value at 52 will now assymptoti-eally track (increase) the relatively stable positive maximum values of curve 74, 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 minima 80). At any time that the amplitude of eurve 74 goes 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 is 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 cireuit.
Thus, electronic switch 48 opens when the signal at 78 is greater than the signal at 79. The maxima of signal 74 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 7 goes below its prior stored value.
The desired positive antigen scatter signal at 52 is summed in an algebraic summinc; amplifier 54 with a suitable negative .
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'107~;3ll6 offset 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 is delivered through a timed switch element 85 to a digital display device S6. The display provided by device 56 is for a selected period of operation of the algebraic sum~ing ampiifier 54, controlled 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. ~t the end of the timed period, switch element 76 opens and closes, producing the digital display in device 56.
The negative antibody blank adjust~ent 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 prlor 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 of algebraic summing amplifier 54, with the antibody blank switch 91 open, in a prior test on a plàin antigen reference sample before antibody - . , ,. , , : :. , , , . ~ , , : . , :
~0,63~6 material is added. This antigen (serum) blank signal is stored in integrator 83 by activating a "set" switch 93 during such a prior test. The 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 abo~e 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 therefore be used to provide an accurate indication of the amount of antigen origïnally present in a sample after the sample has been exposed to a known quantity of antihody 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 56a.
` ` In Figure 6 there are four optical test stations lla to lld. At station lla, a forward scatter measurement is made of ~: 1 : , ,, .. . .:, . .
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the buffer solution, which is available 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 con~ains as ingredients both the antibody and the serum sample, which react to produce the im~unochemicai complex whose concentration is to be indicated at 56a.
The signal at line 101 is one which 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 for the instrument.
The signals at lines 102 to 104 are irregularly affected by dust, and the dust signals are el-iminated by means of the minimum point memory circuits 105 to 107. These circuits correspond to the minimum point memory circuit o Figure 5, comprising inte-grator capacitor 77, diode 49, electronic switch 48 and comparator 53.
The signals from differential amplifiers 108 to 110, when combined 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 54a.
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Figure 7 is a simplified block diagram Oe the invention showing how the objectives are accomplished. No attempt has been made to illustrate the actual circuits, but to sho~ functional characteristics. Proba~ly the best way to describe the operation oE the system is to follow the protocol and explain ~hat ha~pens as the operator adjusts the knobs or puslles the difEcrent buttons.
Yor this purpose the diagram shows, adjacent the approprlate place in th~ circuit, the Label which is af~i~ed to the front panel con-trol which is manipulated by the operator during the test.
A significant point that must ~e remembered for proper understanding is that there are four SPECII~IEN SWITC~I positions, BUFFER BL~NK, I~NTIE30DY BLAN~, S~lPLE BL~NK, and S~IPLE E~EAD, which are mutually e~clusive.
Pro~ocol A. ~fter a 8n minute warm-up, turn E~IGH ~T0LTAGE on. This con-nects negative voltage to the cathode of the photomultiplier tube.
B. Lift sc~mple station cover. PHOTOMETER BLANK switch to MEDIU~I.
~ SE~SITIVITY switch to X3. Adjust analog n:eter to zero using ; the PEIOTO~TER BLANK fine controls. This step establish~s electronlc "zero".
C. Put a tube containing the highest "reference" concentration of antigen/antibody into the sample station and adjust the s~nsitivity controls until the analog meter reads "9" (or any other agreed upol; value). Adjustment of sensitivity insures that all further readings will be in scale.
D. Set SPECI;~lEN SWITCEI to BUE~FER 8L~NK. The digital meter is connected to the output of the variable gain photometer. All other parts o~ tlle circuit are disconnected from the d~ital meter.
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~76386 Insert buffer blank t~lbe in optical station and adjust the value of the digital meter to zero using the PHOTOMETER
BLANK subtract controls. For most immunology experiments the coarse PHOTOMETER BLANK switch will remain set at MEDIUM.
The error contributed by the buffer scatter will now be subtracted from all subsequent readings, taken on other tubes.
F. Adjust ANTIBODY 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 ANTIsoDY
~LANK potentiometer which was set at zero.
iii. Digital 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 biank tube in optical station and pu;h 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 the 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 ~NTIBODY BLANK potentiometer CCW until the digital meter reàds zero. Voltage at pin 2 of final amplifier is increased until it equals the value at the output of the minimum point detector . Since tllat value is the value of : . , : . . , :, 1~176386 the antibody blank, the antibody blank value is permanently recorded in the potentiometer.
J. Set SPECIMEN switch to SA~PLE BLANK. The di~ital meter still reads the output of the final amplifier, the inte~rator is stili set to zero and the ANTIBODY BLANK potentiometer is disabled. The number dlsplayed is the value of the antibody blank obtained in previous steps.
X. Insert the sample blank tube into the optical station and push COMPUTE. The minimum point memory is reset to æero. 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.
. SPECIMEN switch is set to SP~E READ. The digital meter still reads the output of the final amplifier. The action of going to "read" triggers the 0.5 second timer wllich controls contacts Kla and Klb. Contact Xla shorts input #2 of final amplifier for the first 0.5 sec. Contact Klb connects the input of the integrator to the output of the final amplifier; as a result, in?ut #3 of the final amplifier will gradually increase until it is equal to input ~ 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 Rla 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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lU76386 ~1. Insert tube containing 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 minimum point which is fed to input #l 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 ~ETER. Thls is the only nùmber the technician secords.
A prototype or preferred embodiment of the invention has recently been built. This is shown in Figures 8, 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. The latter has a cover 21a which is hinged at the rear edge, unlike the flanged cover 21 Qf 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 oE
Figure 8, and does not have to be manually removed. The photo-multiplier unit, with its high voltage connector projecting to the 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 wiring diagram in ~reater detail than in Figure 7. It will be noted that each of the four SPECIMEN SELECT push buttons and the DAMPING push buttons are self lighting when actuated, thus assisting the operator to keep track of his operations. All of the electronic circuitry is straightforward 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 method and apparatus have been disclosed in the foregoing descrip~ion, it will be understood that various modifications 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 oE the appended claims.
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Claims (8)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A nephelometer for assaying immunochemical complex of a sample and antigen mixture held in a liquid-containing tube, comprising:
means for holding the tube such that its central axis is substantially vertical;
a laser light source for producing a beam of essentially mono-chromatic light, said beam being narrow with respect to the diameter of the tube holding the mixture, and said source being positioned to project the laser beam through the side wall of the tube such that it passes through liquid contained therein at or near the central axis of the tube, and such that the laser beam is at a sub-stantial right angle to the central axis of the tube;
light collecting means for receiving forward scattered light, said light collecting means including a passageway and light shielding means around said passageway for shielding all but light from and about a path through the passageway and through the tube, and said path intersecting the laser light beam in the tube at a point away from the tube walls and lying in a plane which is approximately coplanar with a plane containing the laser beam and the tube axis, and means for sensing the light scattered along the path defined by said light collecting means and for providing an assay output based on the sensed light.
means for holding the tube such that its central axis is substantially vertical;
a laser light source for producing a beam of essentially mono-chromatic light, said beam being narrow with respect to the diameter of the tube holding the mixture, and said source being positioned to project the laser beam through the side wall of the tube such that it passes through liquid contained therein at or near the central axis of the tube, and such that the laser beam is at a sub-stantial right angle to the central axis of the tube;
light collecting means for receiving forward scattered light, said light collecting means including a passageway and light shielding means around said passageway for shielding all but light from and about a path through the passageway and through the tube, and said path intersecting the laser light beam in the tube at a point away from the tube walls and lying in a plane which is approximately coplanar with a plane containing the laser beam and the tube axis, and means for sensing the light scattered along the path defined by said light collecting means and for providing an assay output based on the sensed light.
2. The nephelometer according to claim 1 wherein the path defined by said light collecting means and extending from said laser beam intersects the laser beam at a point within the liquid in the tube and at an angle of approximately 30 degrees thereto.
3. The nephelometer according to claim 2 wherein said holding means includes a cover, and said nephelometer further includes shutter means for blocking the laser beam and preventing same from reaching the tube when said cover is open, said shutter means being operable to automatically move said shutter means to a position blocking the laser beam from reaching the tube when said cover is opened.
4. An improved method of detecting and measuring the quantity of antigens present in a sample by nephelometric means comprising the steps of:
mixing a known quantity of the sample and a selected quantity of an antibody in a tube so as to allow immunocomplex particles to form and orientating the tube containing such mixture and any resulting particles in a generally vertical position with the central axis of the tube oriented in a generally vertical direction;
subjecting the tube and the mixture therein to a narrow beam of laser light of a wavelength greater than, or on the order of, the size of the immunocomplex particles, said beam having a cross-sectional area considerably smaller than the diameter of the tube and being directed through the side wall of the tube at a right angle to the central axis of the tube; and sensing and measuring the quantity of light scattered along a path from the tube defined by a light shielding and collecting apparatus, said path intersecting the laser beam in the mixture in the tube at a position remote from the sidewalls of the tubes and said path lying along a line which extends from the laser beam at a forward scatter angle and which intersects the sidewall of the tube at a point which is not in, or close to, a plane which is perpendicular to the axis of the tube and which includes any portion of the laser beam passing through the tube, whereby receipt of laser light reflected by the walls of the tube is minimized.
mixing a known quantity of the sample and a selected quantity of an antibody in a tube so as to allow immunocomplex particles to form and orientating the tube containing such mixture and any resulting particles in a generally vertical position with the central axis of the tube oriented in a generally vertical direction;
subjecting the tube and the mixture therein to a narrow beam of laser light of a wavelength greater than, or on the order of, the size of the immunocomplex particles, said beam having a cross-sectional area considerably smaller than the diameter of the tube and being directed through the side wall of the tube at a right angle to the central axis of the tube; and sensing and measuring the quantity of light scattered along a path from the tube defined by a light shielding and collecting apparatus, said path intersecting the laser beam in the mixture in the tube at a position remote from the sidewalls of the tubes and said path lying along a line which extends from the laser beam at a forward scatter angle and which intersects the sidewall of the tube at a point which is not in, or close to, a plane which is perpendicular to the axis of the tube and which includes any portion of the laser beam passing through the tube, whereby receipt of laser light reflected by the walls of the tube is minimized.
5. The method according to claim 4 wherein the path is a narrow path which lies in a generally vertical plane formed by the tube axis and the laser beam and which is at an approximate scatter angle given by the expression:
.theta. = arc sine(8.3 x 10 -4 .lambda.) wherein .theta. is the scatter angle in degrees and .lambda. is the wavelength of the laser in nanometers.
.theta. = arc sine(8.3 x 10 -4 .lambda.) wherein .theta. is the scatter angle in degrees and .lambda. is the wavelength of the laser in nanometers.
6. The method according to claim 4 wherein the particles are on the order of 0.2 to 0.4 microns in size, and the path is a narrow path which extends upwardly from the laser beam which emits red light, said path intersecting the laser beam at a point within the mixture in the tube and at an angle of approximately 30 degrees thereto.
7. A nephelometer for assaying immunochemical complex of a sample and antigen mixture held in a liquid-containing tube, comprising:
means for holding the tube such that its central axis is substantially vertical;
a laser light source for producing a beam of essentially mono-chromatic light, said beam being narrow with respect to the diameter of the tube holding the mixture, and said source being positioned to project the laser beam through the side wall of the tube such that it passes through the liquid contained therein along a line intersecting the central axis of the tube and such that the laser beam is at a substantial right angle to the central axis of the tube;
means for collecting light which is forward scattered from said laser beam by complexes in the liquid, said collecting means including means for shielding light from the intersection of the tube walls and the laser beam and for shielding light reflected in a plane which is perpendicular to the axis of the tube and which contains any portion of the laser beam passing through the tube, so that only light from the interior of the tube and out of that plane is unshielded and collected;
means for sensing said unshielded and collected light and for producing and output based upon the sensed light.
means for holding the tube such that its central axis is substantially vertical;
a laser light source for producing a beam of essentially mono-chromatic light, said beam being narrow with respect to the diameter of the tube holding the mixture, and said source being positioned to project the laser beam through the side wall of the tube such that it passes through the liquid contained therein along a line intersecting the central axis of the tube and such that the laser beam is at a substantial right angle to the central axis of the tube;
means for collecting light which is forward scattered from said laser beam by complexes in the liquid, said collecting means including means for shielding light from the intersection of the tube walls and the laser beam and for shielding light reflected in a plane which is perpendicular to the axis of the tube and which contains any portion of the laser beam passing through the tube, so that only light from the interior of the tube and out of that plane is unshielded and collected;
means for sensing said unshielded and collected light and for producing and output based upon the sensed light.
8. A nephelometer for assaying immunochemical complex of a sample and antigen mixture held in a liquid-containing tube, comprising:
a light source for producing a beam of light, said beam being narrow with respect to the diameter of the tube holding the mixture, and said source being positioned to project the beam through the side wall of the tube such that it passes through liquid contained therein at or near the central axis of the tube;
light collecting means for receiving scattered light, said light collecting means including a passageway and light shielding means around said passageway for shielding all but light from and about a path through the passageway and through the tube, and said path intersecting the light beam in the tube at a point away from the tube walls and lying in a plane which is approximately coplanar with a plane containing the beam and the tube axis, and means for sensing the light scattered along the path defined by said light collecting means and for providing an assay output based on the sensed light.
a light source for producing a beam of light, said beam being narrow with respect to the diameter of the tube holding the mixture, and said source being positioned to project the beam through the side wall of the tube such that it passes through liquid contained therein at or near the central axis of the tube;
light collecting means for receiving scattered light, said light collecting means including a passageway and light shielding means around said passageway for shielding all but light from and about a path through the passageway and through the tube, and said path intersecting the light beam in the tube at a point away from the tube walls and lying in a plane which is approximately coplanar with a plane containing the beam and the tube axis, and means for sensing the light scattered along the path defined by said light collecting means and for providing an assay output based on the sensed light.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US54506675A | 1975-01-29 | 1975-01-29 | |
| US60078775A | 1975-07-31 | 1975-07-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1076386A true CA1076386A (en) | 1980-04-29 |
Family
ID=27067814
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA239,752A Expired CA1076386A (en) | 1975-01-29 | 1975-11-17 | Nephelometer having means semiautomatically cancelling components from scattering by particles smaller or larger than those of interest |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1076386A (en) |
-
1975
- 1975-11-17 CA CA239,752A patent/CA1076386A/en not_active Expired
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