IL44675A - Method for the determination of the concentration of lh and a serodiagnostic composition therefor - Google Patents

Description

A the determination of the of ana a Rafa Laboratories Ltd The present Invention relates to a composition for determination of the concentration of the luteinizing hormone called During ovulation increased concentrations of LH are found in the body fluid of the Before ovulation the concentration of the LH in urine is about 2 rises to a peak of about at the time of testing urine samples daily between periods it possible to determine the day on which tho LH concentration rises to its peak and the day of the day on which the woman is She concentration of the LH can be estimated by neans Of an using human chorionic gonadotropin called as because the two hormones of HCO of present makes use of this Erythrocytes sensitized with HCO are agglutinated by an The hemagglutination inhibited the presence of free starting with a predetermined concentration of it the body fluid or in the presence of an equivalent concentration of Bald predetermined concentration is a function of the sensitivity of the Said sensitivity can be adjusted to any desired value by altering the concentration of the It has been found the purpose of the present the reactants should be so adjusted that hemagglutination is inhibited starting with a concentration In application of which the present application is an there described and a composition for determining whether or not a female is said composition consisting essentially coupled to red blood cells by means of as ling agent It now been found that said composition can also be utilised for the determination of the concentration of in the body fluid of a present invention thus consists in a serodiagnostic composition for tho determination of the concentration of tho luteinizing hormone in the body fluid of a woman said composition consisting essentially of human chorionic gonadotropin hormone coupled to red blood by of glutaraldohyde as coupling agent Said cells will be called herein sensitized red blood cells are preferably obtained from they may be obtained also from any other mice or even from human Said composition when being washed with a borate saline solution be stored for quite a long Said period may be lenthened by the addition of certain Said material is if at in an amount of u to preferably about In order to further increase the stability of the diagnostic composition according to the present invention the sensitised blood cells may after having been washed 2a There is known a similar serodiagnostio composition for the determination of the said composition being called the performance of the test with said known composition has serious inter the centrifugated It requires The diluent has to be The time of is A further incubation is A centrifugation has to be The mixture has to be transferred to a second for each test at least two tubes As will be shown hereinafter the test performed with the composition according to the present invention substantially overcomes the above centrifugated Theurine must not be The diluent has not bo be The time required for is only about one No further incubation is No centrifugation has to performed The entire test can be performed in one invention consists also in a method for the determination of the concentration of LH in the body fluid of woman which method comprises admixing said fluid Kith an serum and admixing the admixture obtained with a composition according to the present admixture of the body fluid and the serum is advantageously being left to stand for about 1 hour be fore the serodiagnoatic composition is As body fluid there may be serum or Preferably freshly filtered morning urine is At the beginning and at the end of the menstrual cycle the concentration of the LH in the urine is below that fixed for the sensitivity of the hemagglutination will She moment the predetermined concentration is after about 11 days case that said concentration is 5 no further hemagglutination will occur and a button or ring ion is happens upto about the 20th er the beginning of the Anybody in the performance of the test according to the strength of the determine qualitatively the concentration of the said concentration can be determined quantitatively by diluting the urine and repeating the The on which the concentration of LH in the urine is the highest indicates the day of the day on which the woman is By repeating the tests for several one can ascertain on which day of Whereas in a preferred embodiment of the present invention the method is performed in a advantageously in a solution of pH it may also be performed in another the serodiagnostio composition according to the present invention may be pressed into a be impregnated upon a paper strip or the The so stored composition is then brought in a suitable manner in contact with the serum and the body fluid of the It is readily understood that the amounts of the various parts of the serodiagnostic composition have to be so chosen that the HCG is properly connected to the ooated red It is undesirable to utilise a large excess of any of the various parts of the Under certain when both the tized cells and the serum are they can be mixed together and stored this This admixture is also within the scope of the present In case that such an admixture is the method is performed by admixing the urine with this For performing the method according to the present invention known serum may be as long as it is sufficient specific for the HCG an serum as prepared by the method indicated below and developed in connection with the present invention is being The amount of serum to be utilised is ascertained for each serum as will be shown The present invention will now be illustrated with reference to the following Examples without being limited by 5 of a HCO having a potency of 2500 were in 5 of a phosphate buffer of pH solution obtained was with an equal volume of complete 56 nl solution above potency of the HCO preparation is by no means critical for the success of same applies to all following It is that another buffer solution may utilised as long as it fulfills the She pH range be varied between about to and ml of the above were injected to the foot pad and respectively to a rabbit weighing once a week for consecutive One week after the last set of the rabbit was bled and the serum was separated by centrifugation and stored in a Unspocific were aborbed from the serum by mixing equal volumes of the serum and of normal containing inactivated at for 30 minutes and absorbed on an equal volume of packed washed sheep red blood mixture obtained is called highly A was prepared in the same manner as described in step of Exaraple and of the obtained in step to the foot pad and respectively to a rabbit weighing kg once a month for consecutive after the last set of the rabbit s bled and the was separated by centrifugation and stored in a were absorbed from the serum in the same manner aa described in step in Example Further were absorbed by mixing the with y proteins and same after obtained is called specific absorbed The serum prepared either in Example 1 or in 2 was diluted with to a suitable concentration and finally solution solution g Distilled water at of the 1 of solution in phosphate buffer of pH was added under conetant stirring to the following mixtures phosphate buffer pH 10 packed sheep blood cells the same potency as in a phosphate buffer of pH 2 having a concentration of 12 Stirring was continued or 1 preparation then trashed 3 with a borate salin solution as described and then stored in the is added to the suspension of sensitised advantageously abou of preparation be varied to a the pH of the buffer vary between the solution have a concentration of and the iplutaraldehyde one of In this respect also to 3 in Specification The borate solution was prepared as Sodium Chloride up to of serum Twofold dilutions Borate of were in suitable test each tube having a volume of ml of the as described in added to each test concentration of the blood cello The tubes were shaken and left to stand at room temperature for about At the end of this the results were read and the concentration of the highest dilution of the serum which atill agglutinated in the red was This concentration the end point o the titratio and is defined as For the ovulation test according to the present invention a suitable concentration which vary from and is preferably about ovulation Homing urine samples were collected from women each day of their menstrual serum was dissolved by adding ml of borate Aliquo of ml of the serum solution were distributed into test which were marked according the number of days after the last menstruation from which the urine samples were into 1 control ml of filtered urine was added to respective teat ml solution added to the control tube containing nl of a and of borate saline solution was added to a test The rack containing the test tubes was shaken lightly and left to stand for 1 hour without the influence of outside After 1 hour nl of the sensitised cells prepared as in was added to the respective tube and each of the control She rack containing the test tubes was shaken for 1 and then le to stand 2 hours without vibration and without the influence of outside Finally it observed whether there was or button or ring performed The distribution of the reagents shown in the following Table TOO PERIODS Serum Erythrocyte 9 10 11 12 13 1415 1617 Control Control Urine to Diluent Hemagglutination resembling that observed in the serum control tube indicates that the respective test tube contains less than 50 A button or ring formation to that observed in the erythrocyte control indicates that the tested urine contains at least 50 or quantity of the hormone rises gradually during the first days after menstruation from levels of attains the maximum level of HCO urine and decreases to the initial prior to the next In case that in more than one test tube levels of more than 50 urine are a quantitative test is advantageously performed so that the day of ovulation be more precisely The test is performed by diluting the sensitivity of the teet can be increased to urine by adding of urine to a teet tube containing of of as and by adding of additional diluent to each of control teat In the annexed there are illustrated the of the tests of the period of the menstrual cycle for 2 She on the right aide are the of qualitative whereas the curves on the left side are the of the quantitative Xt apparent that the of ovulation for first vonan is the 14th day after menstruation for the second the 16th day after insufficientOCRQuality

Claims (1)

1. A for tho determination of the of luteinizing in the body fluid of a said composition consisting essentially of human chorionic gonadotropin hormone coupled red blood cells by means of glutaraldehyde as coupling agent Composition according to Claim the red blood cells Composition according to 1 or 2 being suspended in a solution buffered at a pH of between Composition according to Claim wherein said buffering solution is a boric Composition according to any of Claims 1 to wherein said solution contains A composition for the determination of the concentration of the luteinizing the body fluid of a substantially as hereinbefore described with reference to the Method of producing the composition according to Claim 2 or which comprises mixing red blood colls and in a solution of whereby said glutaraldehyde causes coupling of sad to said red blood Method according to Claim wherein said solution buffered at a pH between about 5 and 8 Method according to Claim wherei the buffer solution is a boric Method according to Claim wherein the composition is 13 Method according to Claim wherein said glutaraldehyde or solution contains of producing the composition according to any of 1 to substantially as hereinbefore described with reference to the A for the of tho concentration of LH in fluid of a woman in which said body fluid is admixed with an and the obtained is admixed with composition according to any of 1 to A method according to Claim wherein the body fluid is A according to Claim 13 or therein the of the body fluid and the is left to stand for 1 hour before modification of the od according to 13 or wherein an of eerodiagnostic composition according to any of 1 to 6 and serum admixed with the body A method for the determination of of LH in the body fluid of a substantially as hereinbefore described with to the for tho determination of the day of ovulation which comprises at least during one menstrual dail tests by the method according to any of Claims 13 to determining qualitatively quantitatively the day on which LH concentration is the the day of the Applicants Yitzhak Hess insufficientOCRQuality